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Sample records for controls dectin-1 expression

  1. Selective C-Rel activation via Malt1 controls anti-fungal T(H-17 immunity by dectin-1 and dectin-2.

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    Sonja I Gringhuis

    Full Text Available C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H1 and T(H-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to T(H-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of T(H-17-polarizing cytokines IL-1β and IL-23p19. Malt1 inhibition abrogates c-Rel activation and T(H-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of T(H-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized T(H-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive T(H-17 immunity to fungi via Malt1-dependent activation of c-Rel.

  2. Selective C-Rel activation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin-2.

    Science.gov (United States)

    Gringhuis, Sonja I; Wevers, Brigitte A; Kaptein, Tanja M; van Capel, Toni M M; Theelen, Bart; Boekhout, Teun; de Jong, Esther C; Geijtenbeek, Teunis B H

    2011-01-20

    C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T(H)-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to T(H)-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of T(H)-17-polarizing cytokines IL-1β and IL-23p19. Malt1 inhibition abrogates c-Rel activation and T(H)-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of T(H)-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized T(H)-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive T(H)-17 immunity to fungi via Malt1-dependent activation of c-Rel.

  3. Dectin-1内在化表达介导氧依赖性方式杀伤白色念珠菌%Intracellular expression of Dectin-1 mediates the killing of Candida albicans by human neutrophils in a manner dependent on ROS production

    Institute of Scientific and Technical Information of China (English)

    董碧麟; 李东升; 段逸群; 柳卫凰; 陈金波; 刘伟

    2012-01-01

    目的 探讨Dectin-1介导人嗜中性粒细胞以氧依赖性方式杀伤调理吞噬后白色念珠菌孢子的机制.方法 在白色念珠菌孢子作用前后,流式细胞术和激光共聚焦显微镜检测人嗜中性粒细胞中Dectin-1的表达率及表达部位;并通过Dectin-1阻断试验分析嗜中性粒细胞中Dectin-1的表达与胞内活性氧簇(reactive oxygen species,ROS)的产生及对白色念珠菌孢子杀伤的相关性.结果 在白色念珠菌孢子刺激30或60 min后,细胞内Dectin-1的表达率明显上调(0min,8.32%;30 min,16.82%;60 min,23.88%.与0min相比,P均<0.01),且Dectin-1与ROS在胞内的表达均被募集到吞噬的白色念珠菌孢子表面.Dectin-1的阻断分别与ROS峰值的抑制(R2=0.306,P<0.01)及白色念珠菌杀伤率的降低(R2=0.251,P<0.01)呈剂量依存关系.结论 Dectin-1可通过内在化的表达模式介导人嗜中性粒细胞以ROS依赖性方式杀伤调理吞噬的白色念珠菌孢子.%Objective To investigate the mechanism hy which Dectin-1 mediates the killing of Candida albicans(C.albicans) by human neutrophils in a manner dependent on the production of reactive oxygen species (ROS).Methods After stimulation with FITC-C.albicans at a multiplicity of infection (MOI) of 10 for 30 or 60 min,PE-anti-human Dectin-1 monoclonal antibody (2.5 μg/106 cells) was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry.For Dectin-1 inhibition test and ROS assay,human neutrophils (2×106/ml) were respectively pre-incubated with different concentrations of blocking antibody (0.5,1,2.5 and 5 μg/ml) for 60 min at 4℃,and then with 25 μmol/L 2′,7′-Dichlorofluorescein diacetate for another 20 min at room temperature.Afterwards,under stimulation with live C.albicans at a MOI of 10,the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min.In addition,localization of Dectin-1

  4. Expression of Dectin-1 and Enhanced Activation of NALP3 Inflammasome Are Associated with Resistance to Paracoccidioidomycosis

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    Vera Lucia Garcia Calich

    2015-09-01

    Full Text Available Dectin-1 is a pattern recognition receptor (PRR that recognizes β-glucans and plays a major role in the immunity against fungal pathogens. Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, has a sugar-rich cell wall mainly composed of mannans and glucans. To investigate the role of dectin-1 in the innate immunity of resistant (A/J and susceptible (B10.A mice to P. brasiliensis infection, we evaluated the role of curdlan (a dectin-1 agonist and laminarin (a dectin-1 antagonist in the activation of macrophages from both mouse strains. We verified that curdlan has a negligible role in the activation of B10.A macrophages but enhances the phagocytic and fungicidal abilities of A/J macrophages. Curdlan up-regulated the expression of costimulatory molecules and PRRs in A/J macrophages that express elevated levels of dectin-1, but not in B10.A cells. In addition, curdlan treatment inhibited arginase-1 and enhanced NO-synthase mRNA expression in infected A/J macrophages but had not effect in B10.A cells. In contrast, laminarin reinforced the respective M2/M1 profiles of infected A/J and B10.A macrophages. Following curdlan treatment, A/J macrophages showed significantly higher Syk kinase phosphorylation and expression of intracellular pro-IL-1β . These findings led us to investigate if the NRLP3 inflammasome was differently activated in A/J and B10.A cells. Indeed, compared with B10.A cells A/J macrophages showed an increased expression of NALP3, ASC and IL-1β mRNA. They also showed elevated caspase-1 activity and secreted high levels of mature IL-β and IL-18 after curdlan treatment and P. brasiliensis infection. Our data demonstrate that soluble and particulate β-glucans exert opposed modulatory activities on macrophages of diverse genetic patterns. Moreover, the synergistic action of dectin-1 and NALP3 inflammasome were for the first time associated with the innate

  5. Different expression of dectin-1 and Toll-like receptor 2 in the lungs of different immune status mice infected with Aspergillus fumigatus

    Institute of Scientific and Technical Information of China (English)

    YANG Jian-xun; LIU Wan; LU Qiao-yun; WAN Zhe; WANG Xiao-hong; LI Ruo-yu

    2009-01-01

    would provide a broad range of possibilities for a specific and effective inflammatory response to kill A. fumigatus. Inhibition of dectin-1 expression may be one of the mechanisms of cyclophosphamide in the development of IPA.

  6. G-test positive serum inhibits ROS-dependent killing of Candida albicans by interfering with internal- ized expression of neutrophil Dectin-1%G 试验阳性血清通过干扰 Dectin-1的内在化表达抑制 ROS 依赖性杀伤白念珠菌

    Institute of Scientific and Technical Information of China (English)

    董碧麟; 汪明; 柳卫凰; 李东升; 童中胜; 段逸群; 刘伟

    2013-01-01

    Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0

  7. Fasciola hepatica excretory-secretory products induce CD4+T cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way.

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    Guasconi, Lorena; Chiapello, Laura S; Masih, Diana T

    2015-07-01

    Fasciola hepatica excretory-secretory products (FhESP) induce immunomodulatory effects on macrophages. Previously, we demonstrated that these effects are dependent on Dectin-1. Therefore, the aim of this study was to determine how this affects the CD4 T-cells immune response. We observed that FhESP induce an increased expression of PD-L2 in macrophages via Dectin-1. Furthermore, in co-cultures with CD4 T-cell we observed a suppressive effect on proliferative response, down-modulation of IFN-γ and up-modulation of IL-10 via Dectin-1 on macrophages. These results suggest that FhESP induce T-cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way.

  8. Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways.

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    Seifert, Lena; Deutsch, Michael; Alothman, Sara; Alqunaibit, Dalia; Werba, Gregor; Pansari, Mridul; Pergamo, Matthew; Ochi, Atsuo; Torres-Hernandez, Alejandro; Levie, Elliot; Tippens, Daniel; Greco, Stephanie H; Tiwari, Shaun; Ly, Nancy Ngoc Giao; Eisenthal, Andrew; van Heerden, Eliza; Avanzi, Antonina; Barilla, Rocky; Zambirinis, Constantinos P; Rendon, Mauricio; Daley, Donnele; Pachter, H Leon; Hajdu, Cristina; Miller, George

    2015-12-01

    Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways

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    Lena Seifert

    2015-12-01

    Full Text Available Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1–/– mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

  10. Human Dectin-1 Deficiency and Mucocutaneous Fungal Infections

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    Ferwerda, Bart; Ferwerda, Gerben; Plantinga, Theo S.; Willment, Janet A.; van Spriel, Annemiek B.; Venselaar, Hanka; Elbers, Clara C.; Johnson, Melissa D.; Cambi, Alessandra; Huysamen, Cristal; Jacobs, Liesbeth; Jansen, Trees; Verheijen, Karlijn; Masthoff, Laury; Morré, Servaas A.; Vriend, Gert; Williams, David L.; Perfect, John R.; Joosten, Leo A.B.; Wijmenga, Cisca; van der Meer, Jos W.M.; Adema, Gosse J.; Kullberg, Bart Jan; Brown, Gordon D.; Netea, Mihai G.

    2009-01-01

    SUMMARY Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the β-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate β-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with β-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense. PMID:19864674

  11. Selective C-Rel Activation via Malt1 Controls Anti-Fungal T-H-17 Immunity by Dectin-1 and Dectin-2

    NARCIS (Netherlands)

    Gringhuis, S.I.; Wevers, B.A.; Kaptein, T.M.; Capel, T.M.M.; Theelen, B.; Boekhout, T.; de Jong, E.C.; Geijtenbeek, T.B.H.

    2011-01-01

    C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T-H-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-kappa B. Here we demonstr

  12. Selective C-Rel activation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin-2

    NARCIS (Netherlands)

    Gringhuis, S.I.; Wevers, B.A.; Kaptein, T.M.; van Capel, T.M.; Theelen, B.J.F.; Boekhout, T.; de Jong, E.C.; Geijtenbeek, T.B.H.

    2011-01-01

    C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T(H)-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-kappaB. Here we demonstr

  13. Dectin-1 and Dectin-2 in innate immunity against fungi.

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    Saijo, Shinobu; Iwakura, Yoichiro

    2011-08-01

    Dectin-1 and Dectin-2 are type II transmembrane proteins of the C-type lectin family with single carbohydrate recognition domains (CRDs) in their extracellular region. They are expressed mainly in dendritic cells and macrophages. Dectin-1 recognizes β-glucans with its CRD and transduces signals through its immunoreceptor tyrosine-based activation motif (ITAM)-like motif in the cytoplasmic domain, whereas Dectin-2 recognizes α-mannans and transduces its signal through association with the ITAM-containing Fc receptor γ chain. Upon ligand binding, spleen tyrosine kinase is recruited to the ITAM and activates the caspase recruitment domain family member 9 (CARD9)-nuclear factor-κB axis, resulting in the activation of various genes including those encoding pro-inflammatory cytokines. Both β-glucans and α-mannans are major cell wall components of fungi including Candida albicans and Pneumocystis carinii. Recently, it was reported that Dectin-1 is important in protection against P. carinii by inducing reactive oxygen species, whereas both Dectin-1 and Dectin-2 play important roles in defense against C. albicans by preferentially inducing T(h)17 cell differentiation. In this review, we briefly revisit the structures, ligands, signal transduction and functional roles of Dectin-1 and Dectin-2 in host defense against fungal infection.

  14. Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1

    NARCIS (Netherlands)

    Marakalala, M.J.; Vautier, S.; Potrykus, J.; Walker, L.A.; Shepardson, K.M.; Hopke, A.; Mora-Montes, H.M.; Kerrigan, A.; Netea, M.G.; Murray, G.I.; MacCallum, D.M.; Wheeler, R.; Munro, C.A.; Gow, N.A.; Cramer, R.A.; Brown, A.J.; Brown, G.D.

    2013-01-01

    The beta-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fun

  15. Genetic deletion of dectin-1 does not affect the course of murine experimental colitis

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    Heinsbroek Sigrid EM

    2012-04-01

    Full Text Available Abstract Background It is believed that inflammatory bowel diseases (IBD result from an imbalance in the intestinal immune response towards the luminal microbiome. Dectin-1 is a widely expressed pattern recognition receptor that recognizes fungi and upon recognition it mediates cytokine responses and skewing of the adaptive immune system. Hence, dectin-1 may be involved in the pathogenesis of IBD. Methods We assessed the responses of dectin-1 deficient macrophages to the intestinal microbiota and determined the course of acute DSS and chronic Helicobacter hepaticus induced colitis in dectin-1 deficient mice. Results We show that the mouse intestinal microbiota contains fungi and the cytokine responses towards this microbiota were significantly reduced in dectin-1 deficient macrophages. However, in two different colitis models no significant differences in the course of inflammation were found in dectin-1 deficient mice compared to wild type mice. Conclusions Together our data suggest that, although at the immune cell level there is a difference in response towards the intestinal flora in dectin-1 deficient macrophages, during intestinal inflammation this response seems to be redundant since dectin-1 deficiency in mice does not affect intestinal inflammation in experimental colitis.

  16. Enhanced immunogenicity of a tricomponent mannan tetanus toxoid conjugate vaccine targeted to dendritic cells via Dectin-1 by incorporating β-glucan.

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    Lipinski, Tomasz; Fitieh, Amira; St Pierre, Joëlle; Ostergaard, Hanne L; Bundle, David R; Touret, Nicolas

    2013-04-15

    In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the β-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the β-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the β-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-β and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans β-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.

  17. Dectin-1 agonist selectively induces IgG1 class switching by LPS-activated mouse B cells.

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    Seo, Beom-Seok; Park, Ha-Yan; Yoon, Hee-Kyung; Yoo, Yung-Choon; Lee, Junglim; Park, Seok-Rae

    2016-10-01

    Heat-killed Saccharomyces cerevisiae (HKSC) is an agonist for Dectin-1, a major fungal cell wall β-glucan receptor. We previously reported that HKSC selectively enhances IgG1 production by LPS-activated mouse B cells. To determine if this IgG1 selectivity is caused by selective IgG1 class switching, we performed RT-PCRs for measuring germline transcripts (GLTs), flow cytometric analyses for detecting Ig-expressing cells, and ELISPOT assays for measuring the number of Ig-secreting cells in HKSC/LPS-stimulated mouse B cell cultures. HKSC selectively enhanced expression of GLTγ1, the number of IgG1-expressing cells, and the number of IgG1-secreting B cells in the presence of LPS stimulation. In addition, HKSC induced the expression of CD69, an activation marker for B lymphocytes, and the expression of surface Dectin-1. Two Dectin-1 antagonists, laminarin and a neutralizing Dectin-1 antibody, selectively diminished HKSC-reinforced IgG1 production by LPS-stimulated B cells. Furthermore, depleted zymosan (dzn), a Dectin-1 agonist with increased selectivity, also selectively enhanced GLTγ1 transcription. The Dectin-1 antagonists blocked dzn-induced IgG1 production by LPS-activated B cells. Collectively, these results suggest that Dectin-1 agonists selectively induce IgG1 class switching by direct stimulation of Dectin-1 on LPS-activated B cells resulting in selective production of IgG1.

  18. Distinct roles for Dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus keratitis.

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    Sixto M Leal

    Full Text Available Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP-expressing A. fumigatus (Af293.1RFP conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1beta and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that beta-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1(-/- corneas have impaired IL-1beta and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high beta-glucan. In contrast to Dectin 1(-/- mice, cellular infiltration into infected TLR2(-/-, TLR4(-/-, and MD-2(-/- mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4(-/- mice, but not TLR2(-/- or MD-2(-/- mice. We also found that TRIF(-/- and TIRAP(-/- mice exhibited no fungal-killing defects, but that MyD88(-/- and IL-1R1(-/- mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which beta-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1beta, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4

  19. Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Cheng-Cheng; Zhu; Gui-Qiu; Zhao; Jing; Lin; Li-Ting; Hu; Qiang; Xu; Xu-Dong; Peng; Xue; Wang; Sheng; Qiu

    2015-01-01

    · AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P <0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P <0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

  20. Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

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    Cheng-Cheng Zhu

    2015-08-01

    Full Text Available AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus (A. fumigatus in cultured human corneal epithelial cells (HCECs, and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.METHODS:The HCECs were stimulated by curdlan in different concentrations (50, 100, 200, 400 μg/mL for various time. Then HCECs pretreated with or without laminarin (Dectin-1 blocker, 0.3 mg/mL and curdlan were stimulated by A. fumigatus hyphae. The mRNA and protein production of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were determined by real-timequantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.RESULTS: Curdlan stimulated mRNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at mRNA and protein levels compared with A. fumigatus hyphae stimulation group (P<0.05. Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1 expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphaestimulation. The Dectin-1 blocker laminarin suppressed the mRNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae (P<0.05.CONCLUSION:These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs. Dectin-1 is essential for the immunomodulatory effects of curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

  1. Fungal recognition enhances mannose receptor shedding through dectin-1 engagement.

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    Gazi, Umut; Rosas, Marcela; Singh, Sonali; Heinsbroek, Sigrid; Haq, Imran; Johnson, Simon; Brown, Gordon D; Williams, David L; Taylor, Philip R; Martinez-Pomares, Luisa

    2011-03-11

    The mannose receptor (MR) is an endocytic type I membrane molecule with a broad ligand specificity that is involved in both hemostasis and pathogen recognition. Membrane-anchored MR is cleaved by a metalloproteinase into functional soluble MR (sMR) composed of the extracellular domains of intact MR. Although sMR production was initially considered a constitutive process, enhanced MR shedding has been observed in response to the fungal pathogen Pneumocystis carinii. In this work, we have investigated the mechanism mediating enhanced MR shedding in response to fungi. We show that other fungal species, including Candida albicans and Aspergillus fumigatus, together with zymosan, a preparation of the cell wall of Saccharomyces cerevisiae, mimic the effect of P. carinii on sMR production and that this effect takes place mainly through β-glucan recognition. Additionally, we demonstrate that MR cleavage in response to C. albicans and bioactive particulate β-glucan requires expression of dectin-1. Our data, obtained using specific inhibitors, are consistent with the canonical Syk-mediated pathway triggered by dectin-1 being mainly responsible for inducing MR shedding, with Raf-1 being partially involved. As in the case of steady-state conditions, MR shedding in response to C. albicans and β-glucan particles requires metalloprotease activity. The induction of MR shedding by dectin-1 has clear implications for the role of MR in fungal recognition, as sMR was previously shown to retain the ability to bind fungal pathogens and can interact with numerous host molecules, including lysosomal hydrolases. Thus, MR cleavage could also impact on the magnitude of inflammation during fungal infection.

  2. Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-α production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1.

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    Pedroza, Luis A; Kumar, Vipul; Sanborn, Keri B; Mace, Emily M; Niinikoski, Harri; Nadeau, Kari; Vasconcelos, Dewton de Moraes; Perez, Elena; Jyonouchi, Soma; Jyonouchi, Harumi; Banerjee, Pinaki P; Ruuskanen, Olli; Condino-Neto, Antonio; Orange, Jordan S

    2012-02-01

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of β-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  3. Dectin-1 and Dectin-2 in innate immunity against fungal infection%Dectin-1和Dectin-2在抗真菌免疫中的作用

    Institute of Scientific and Technical Information of China (English)

    孙禾; 徐小勇; 苏欣; 施毅

    2013-01-01

    Dectin-1 and Dectin-2 are type Ⅱ transmembrane proteins of the C-type lectin family with single carbohydrate recognition domains (CRD) in the extracellular region,which are mainly expressed in dendritic cells and macrophages.Dectin-1 recognizes beta-glucans with its CRD and transduces signals through its immunoreceptor tyrosine-based activation motif (ITAM)-like motif in the cytoplasmic domain,while Dectin-2 recognizes alpha-mannans and transduces its signal through association with the ITAM-containing Fc receptor gamma chain.Upon ligand binding,spleen tyrosine kinase is recruited to the ITAM and activates the caspase recruitment domain family member 9 (CARD9)-nuclear factor-kappa B axis,resulting in the activation of various genes including those encoding proinflammatory cytokines.Dectin-1 is important in protection against fungal infection by inducing reactive oxygen species,whereas both Dectin-1 and Dectin-2 play important roles in defense against fungi by preferentially inducing T(h)17 cell differentiation.%Dectin-1和Dectin-2是Ⅱ型跨膜蛋白,属C型凝集素受体家族,在胞外区有独立的糖结合域,主要在树突状细胞和巨噬细胞表面表达.Dectin-1识别真菌细胞壁上的β-葡聚糖,依赖胞浆内的酪氨酸激活基序(ITAM样基序)传递信号;Dectin-2识别α-甘露聚糖,通过胞内的补体FC段的γ链结合ITAM样基序传递信号.激活后的Dectin-1和Dectin-2募集脾脏酪氨酸激酶与酪氨酸激活基序(ITAM基序)结合,并激活CARD9-NF-κB信号通路,导致促炎因子等多种基因表达.Dectin-1诱导真菌感染宿主产生活性氧,在免疫防御中发挥重要作用.Dectin-1和Dectin-2均优先诱导Th17细胞分化,对真菌感染宿主起保护作用.

  4. Biaryl amide compounds reduce the inflammatory response in macrophages by regulating Dectin-1.

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    Hyung, Kyeong Eun; Lee, Mi Ji; Lee, Yun-Jung; Lee, Do Ik; Min, Hye Young; Park, So-Young; Min, Kyung Hoon; Hwang, Kwang Woo

    2016-03-01

    Macrophages are archetypal innate immune cells that play crucial roles in the recognition and phagocytosis of invading pathogens, which they identify using pattern recognition receptors (PRRs). Dectin-1 is essential for antifungal immune responses, recognizing the fungal cellular component β-glucan, and its role as a PRR has been of increasing interest. Previously, we discovered and characterized a novel biaryl amide compound, MPS 03, capable of inhibiting macrophage phagocytosis of zymosan. Therefore, in this study we aimed to identify other biaryl amide compounds with greater effectiveness than MPS 03, and elucidate their cellular mechanisms. Several MPS 03 derivatives were screened, four of which reduced zymosan phagocytosis in a similar manner to MPS 03. To establish whether such phagocytosis inhibition influenced the production of inflammatory mediators, pro-inflammatory cytokine and nitric oxide (NO) levels were measured. The production of TNF-α, IL-6, IL-12, and NO was significantly reduced in a dose-dependent manner. Moreover, the inflammation-associated MAPK signaling pathway was also affected by biaryl amide compounds. To investigate the underlying cellular mechanism, PRR expression was measured. MPS 03 and its derivatives were found to inhibit zymosan phagocytosis by decreasing Dectin-1 expression. Furthermore, when macrophages were stimulated by zymosan after pretreatment with biaryl amide compounds, downstream transcription factors such as NFAT, AP-1, and NF-κB were downregulated. In conclusion, biaryl amide compounds reduce zymosan-induced inflammatory responses by downregulating Dectin-1 expression. Therefore, such compounds could be used to inhibit Dectin-1 in immunological experiments and possibly regulate excessive inflammatory responses.

  5. Innate immunity induced by fungal β-glucans via dectin-1 signaling pathway.

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    Lee, Dong Hee; Kim, Ha Won

    2014-01-01

    Mushrooms are a highly valuable source of substances that possess unique biological properties and medicinal efficacy. Medicinal mushrooms traditionally have been used to treat cancer, fungal infections, hypertension, diabetes, inflammation, and renal disorders. Medicinal mushrooms produce high-molecular-weight β-glucans, which have antitumor and antifungal activities that stimulate innate immunity. Innate immune cells express pattern recognition receptors (PRRs) such as dectin-1, Toll-like receptors, and mannose receptors on their cell surfaces. These PRRs recognize pathogens by binding to highly conserved pathogen-associated molecular patterns such as β-glucan, mannan, and lipopolysaccharide. The immunomodulating activities of innate immune cells are augmented by the binding of β-glucans to dectin-1 that is expressed by macrophages or dendritic cells. Upon binding β-glucan, innate immune cells activate adaptive immune cells such as B and T lymphocytes or natural killer cells by secreting various cytokines such as interleukins (IL-4, IL-6) and tumor necrosis factor-α. Water-insoluble β-glucans have stronger immunostimulating activities than their water-soluble counterparts. β-glucans have antifungal activity that is similar to their anticancer activities and is mediated by binding to dectin-1, albeit by an unknown mechanism. In this review we discuss recent progress in understanding the mechanisms responsible for the antitumor activities of fungal β-glucans that act through pathogen-associated molecular patterns and PRRs.

  6. Polymorphisms of Dectin-1 and TLR2 Predispose to Invasive Fungal Disease in Patients with Acute Myeloid Leukemia.

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    Mike Fischer

    Full Text Available Patients with acute myeloid leukemia (AML who undergo induction chemotherapy are at high risk for invasive fungal disease (IFD. Dectin-1, a C-type lectin family member represents one of the most important pattern recognition receptors of the innate immune system and single nucleotide polymorphisms (SNPs in the Dectin-1 gene have been associated with an increased risk of infectious complications. We sought to investigate the impact of three different Dectin-1 SNPs and one TLR2 SNP on developing IFD in 186 adult patients with newly diagnosed AML following anthracycline-based induction chemotherapy.Genotyping of Dectin-1 SNPs (rs16910526, rs3901533 and rs7309123 and TLR2 SNP (rs5743708 was performed by TaqMan method and pyrosequencing. IFD was defined according to the EORTC/MSG consensus guidelines. Multiple logistic regression analyses were applied to evaluate the association between the polymorphisms and the occurrence of pulmonary infections. Dectin-1 expression studies with SNP genotyped human monocytes were performed to elucidate susceptibility to IFD following chemotherapy.We could demonstrate that patients carrying the Dectin-1 SNP rs7309123 G/G (n = 47 or G/G and C/G (n = 133 genotype revealed a significant higher risk for developing both pneumonia in general (adjusted odds ratio (OR: 2.5; p = 0.014 and OR: 3.0, p = 0.004 and pulmonary IFD (OR: 2.6; p = 0.012 and OR: 2.4, p = 0.041, respectively. Patients carrying the TLR2 SNP rs5743708 (R753Q, GA/AA genotype, n = 12 also revealed a significantly higher susceptibility to pneumonia including IFD. Furthermore, Dectin-1 mRNA expression in human monocytes was lower following chemotherapy.To our best knowledge, this study represents the first analysis demonstrating that harbouring polymorphisms of Dectin-1 (rs7309123 or TLR2 (rs5743708 represents an independent risk factor of developing IFD in patients with AML undergoing induction chemotherapy.

  7. Dectin-1 regulates IL-10 production via a MSK1/2 and CREB dependent pathway and promotes the induction of regulatory macrophage markers.

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    Suzanne E Elcombe

    Full Text Available In response to infection by fungal pathogens, the innate immune system recognises specific fungal pathogen associated molecular patterns (PAMPs via pattern recognition receptors including the C-type lectin dectin-1 and members of the Toll Like Receptor (TLR family. Stimulation of these receptors leads to the induction of both pro- and anti-inflammatory cytokines. The protein kinases MSK1 and 2 are known to be important in limiting inflammatory cytokine production by macrophages in response to the TLR4 agonist LPS. In this study we show that MSKs are also activated in macrophages by the fungal derived ligand zymosan, as well as the dectin-1 specific agonists curdlan and depleted zymosan, via the ERK1/2 and p38α MAPK pathways. Furthermore, we show that MSKs regulate dectin-1 induced IL-10 production, and that this regulation is dependent on the ability of MSKs to phosphorylate the transcription factor CREB. IL-10 secreted in response to zymosan was able to promote STAT3 phosphorylation via an autocrine feedback loop. Consistent with the decreased IL-10 secretion in MSK1/2 knockout macrophages, these cells also had decreased STAT3 tyrosine phosphorylation relative to wild type controls after stimulation with zymosan. We further show that the reduction in IL-10 production in the MSK1/2 macrophages results in increased secretion of IL-12p40 in response to zymosan relative to wild type controls. The production of high levels of IL-10 but low levels of IL-12 has previously been associated with an M2b or 'regulatory' macrophage phenotype, which was initially described in macrophages stimulated with a combination of immune complexes and LPS. We found that zymosan, via dectin-1 activation, also leads to the expression of SphK1 and LIGHT, markers of a regulatory like phenotype in mouse macrophages. The expression of these makers was further reinforced by the high level of IL-10 secreted in response to zymosan stimulation.

  8. Borrelia-induced cytokine production is mediated by spleen tyrosine kinase (Syk) but is Dectin-1 and Dectin-2 independent.

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    Oosting, Marije; Buffen, Kathrin; Cheng, Shih-Chin; Verschueren, Ineke C; Koentgen, Frank; van de Veerdonk, Frank L; Netea, Mihai G; Joosten, Leo A B

    2015-12-01

    Although it is known that Borrelia species express sugar-like structures on their outer surface, not much is known about the role of these structures in immune recognition by host cells. Fungi, like Candida albicans, are mainly recognized by C-type lectin receptors, in specific Dectin-1 and Dectin-2. In this study we assessed the role of Dectin-1 and Dectin-2 in the recognition process of Borrelia spirochetes. Using specific inhibitors against these receptors on human cells did not influenced cytokine production. Individuals carrying a SNP leading to an early stop codon in the DECTIN-1 gene also did not lead to differential induction of Borrelia-dependent cytokines. After injection of live Borrelia into knee joints of Dectin-2 deficient mice a trend towards lower inflammation was observed. Inhibition of Syk in human cells resulted in lower cytokine production after Borrelia stimulation. In conclusion, Dectin-1 and Dectin-2 seem not to play a major role in Borrelia recognition or Borrelia-induced inflammation. However, Syk seems to be involved in Borrelia-induced cytokine production.

  9. 1,25(OH)2D3 and VDR Signaling Pathways Regulate the Inhibition of Dectin-1 Caused by Cyclosporine A in Response to Aspergillus Fumigatus in Human Corneal Epithelial Cells

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    Xia, Yiping; Zhao, Guiqiu; Lin, Jing; Li, Cui; Cong, Lin; Jiang, Nan; Xu, Qiang; Wang, Qian

    2016-01-01

    Background The objective of this study is to observe whether cyclosporine A (CsA) inhibits the expression of dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (A. fumigatus) and to investigate the molecular mechanisms of the inhibition. Methods Immortalized human corneal epithelial cells (HCECs) were pretreated with 1,25(OH)2D3 and VDR inhibitor for 1 h, and then they were pretreated with CsA for 12h. After these pretreatments, the HCECs were stimulated with A. fumigatus and curdlan respectively, and the expression of dectin-1 and proinflammatory cytokines (IL-1β and TNF-α) were detected by RT-PCR, western blot and ELISA. Results Dectin-1 mRNA and dectin-1 protein expression increased when HCECs were stimulated with A. fumigatus or curdlan, and CsA inhibited the dectin-1 expression both in mRNA and protein levels specifically. Dectin-1 and proinflammatory cytokine expression levels were higher when HCECs were pretreated with VDR inhibitor and CsA compared to pretreatment with CsA alone, while dectin-1 and proinflammatory cytokine levels were lower when HCECs were pretreated with 1,25(OH)2D3 and CsA compared to pretreatment with CsA alone. Conclusions These data provide evidence that CsA can inhibit the expression of dectin-1 and proinflammatory cytokines through dectin-1 when HCECs are stimulated by A. fumigatus or curdlan. The active form of vitamin D, 1,25(OH)2D3, and VDR signaling pathway regulate the inhibition of CsA. The inhibition is enhanced by 1,25(OH)2D3, and the VDR inhibitor suppresses the inhibition. PMID:27755569

  10. Anion Exchanger 2 Regulates Dectin-1-Dependent Phagocytosis and Killing of Candida albicans.

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    Katia Urso

    Full Text Available Anion exchanger 2 (Ae2; gene symbol, Slc4a2 is a plasma membrane Cl-/HCO3- exchanger expressed in the gastrointestinal tract, kidney and bone. We have previously shown that Ae2 is required for the function of osteoclasts, bone resorbing cells of the macrophage lineage, to maintain homeostatic cytoplasmic pH and electroneutrality during acid secretion. Macrophages require endosomal acidification for pathogen killing during the process known as phagocytosis. Chloride is thought to be the principal ion responsible for maintaining electroneutrality during organelle acidification, but whether Cl-/HCO3- exchangers such as Ae2 contribute to macrophage function is not known. In this study we investigated the role of Ae2 in primary macrophages during phagocytosis. We find that Ae2 is expressed in macrophages where it regulates intracellular pH and the binding of Zymosan, a fungal cell wall derivative. Surprisingly, the transcription and surface expression of Dectin-1, the major phagocytic receptor for Candida albicans (C. albicans and Zymosan, is reduced in the absence of Ae2. As a consequence, Zymosan-induced Tnfα expression is also impaired in Ae2-deficient macrophages. Similar to Ae2 deficiency, pharmacological alkalinization of lysosomal pH with bafilomycin A decreases both Dectin-1 mRNA and cell surface expression. Finally, Ae2-deficient macrophages demonstrate defective phagocytosis and killing of the human pathogenic fungus C. albicans. Our results strongly suggest that Ae2 is a critical factor in the innate response to C. albicans. This study represents an important contribution to a better understanding of how Dectin-1 expression and fungal clearance is regulated.

  11. Dectin-1 and IL-17A suppress murine asthma induced by Aspergillus versicolor but not Cladosporium cladosporioides due to differences in β-glucan surface exposure.

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    Mintz-Cole, Rachael A; Gibson, Aaron M; Bass, Stacey A; Budelsky, Alison L; Reponen, Tiina; Hershey, Gurjit K Khurana

    2012-10-01

    There is considerable evidence supporting a role for mold exposure in the pathogenesis and expression of childhood asthma. Aspergillus versicolor and Cladosporium cladosporioides are common molds that have been implicated in asthma. In a model of mold-induced asthma, mice were repeatedly exposed to either A. versicolor or C. cladosporioides spores. The two molds induced distinct phenotypes, and this effect was observed in both BALB/c and C57BL/6 strains. C. cladosporioides induced robust airway hyperresponsiveness (AHR), eosinophilia, and a predominately Th2 response, whereas A. versicolor induced a strong Th17 response and neutrophilic inflammation, but very mild AHR. Neutralization of IL-17A resulted in strong AHR and eosinophilic inflammation following A. versicolor exposure. In Dectin-1-deficient mice, A. versicolor exposure resulted in markedly attenuated IL-17A and robust AHR compared with wild-type mice. In contrast, C. cladosporioides induced AHR and eosinophilic inflammation independent of IL-17A and Dectin-1. A. versicolor, but not C. cladosporioides, spores had increased exposure of β-glucans on their surface and were able to bind Dectin-1. Thus, the host response to C. cladosporioides was IL-17A- and Dectin-1-independent, whereas Dectin-1- and IL-17A-dependent pathways were protective against the development of asthma after exposure to A. versicolor.

  12. Oxidative and nitric oxide responses in carp macrophages induced by zymosan, MacroGard and selective dectin-1 agonists suggest recognition by multiple pattern recognition receptors

    NARCIS (Netherlands)

    Pietretti, D.; Vera-Jimenez, N.I.; Hoole, D.; Wiegertjes, G.F.

    2013-01-01

    ß-Glucans are glucose polymers that are found in the cell walls of plants, bacteria, certain fungi, mushrooms and the cell wall of baker's yeast. In mammals, myeloid cells express several receptors capable of recognizing ß-glucans, with the C-type lectin receptor dectin-1 in conjunction with

  13. Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia

    NARCIS (Netherlands)

    Rosentul-Amram, D.C.; Plantinga, T.S.; Oosting, M.; Scott, W.K.; Velez Edwards, D.R.; Smith, P.B.; Alexander, B.D.; Yang, J.C.; Laird, G.M.; Joosten, L.A.B.; Meer, J.W.M. van der; Perfect, J.R.; Kullberg, B.J.; Netea, M.G.; Johnson, M.D.

    2011-01-01

    BACKGROUND: Candidemia is an important cause of morbidity and mortality in critically ill patients or patients undergoing invasive treatments. Dectin-1 is the main beta-glucan receptor, and patients with a complete deficiency of either dectin-1 or its adaptor molecule CARD9 display persistent

  14. Differential dependencies of monocytes and neutrophils on dectin-1, dectin-2 and complement for the recognition of fungal particles in inflammation.

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    Jacqueline U McDonald

    Full Text Available We have re-investigated the role of the complement system and the non-opsonic pattern recognition receptors dectin-1 and dectin-2 in the recognition of fungal particles by inflammatory neutrophils, monocytes and macrophages. We have used in vivo and ex vivo models to study the recognition and response of these cells: i We confirm previous observations regarding the importance of complement to neutrophil but not monocytic responses; ii We show that dectin-1 is important for driving inflammatory cell recruitment to fungal stimuli and that it biases the immediate inflammatory response to one that favors neutrophil over monocyte recruitment; iii We show that dectin-2 contributes to the physical recognition of fungal particles by inflammatory monocytes/macrophages, but is also expressed on neutrophils, where we show it has the potential to contribute to cellular activation; iv Additionally, we show that serum-opsonization has the potential to interfere with non-opsonic recognition of fungal particles by dectin-1 and dectin-2, presumably through masking of ligands. Collectively these roles are consistent with previously described roles of dectin-1 and dectin-2 in driving inflammatory and adaptive immune responses and complement in containing fungal burdens. This study emphasizes the importance of heterogeneity of receptor expression across myeloid cell subsets in protective immune responses.

  15. Differential dependencies of monocytes and neutrophils on dectin-1, dectin-2 and complement for the recognition of fungal particles in inflammation.

    Science.gov (United States)

    McDonald, Jacqueline U; Rosas, Marcela; Brown, Gordon D; Jones, Simon A; Taylor, Philip R

    2012-01-01

    We have re-investigated the role of the complement system and the non-opsonic pattern recognition receptors dectin-1 and dectin-2 in the recognition of fungal particles by inflammatory neutrophils, monocytes and macrophages. We have used in vivo and ex vivo models to study the recognition and response of these cells: i) We confirm previous observations regarding the importance of complement to neutrophil but not monocytic responses; ii) We show that dectin-1 is important for driving inflammatory cell recruitment to fungal stimuli and that it biases the immediate inflammatory response to one that favors neutrophil over monocyte recruitment; iii) We show that dectin-2 contributes to the physical recognition of fungal particles by inflammatory monocytes/macrophages, but is also expressed on neutrophils, where we show it has the potential to contribute to cellular activation; iv) Additionally, we show that serum-opsonization has the potential to interfere with non-opsonic recognition of fungal particles by dectin-1 and dectin-2, presumably through masking of ligands. Collectively these roles are consistent with previously described roles of dectin-1 and dectin-2 in driving inflammatory and adaptive immune responses and complement in containing fungal burdens. This study emphasizes the importance of heterogeneity of receptor expression across myeloid cell subsets in protective immune responses.

  16. The Prefrontal Dectin-1/AMPA Receptor Signaling Pathway Mediates The Robust and Prolonged Antidepressant Effect of Proteo-β-Glucan from Maitake

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    Bao, Hongkun; Ran, Pengzhan; Zhu, Ming; Sun, Lijuan; Li, Bai; Hou, Yangyang; Nie, Jun; Shan, Liping; Li, Hongliang; Zheng, Shangyong; Xu, Xiufeng; Xiao, Chunjie; Du, Jing

    2016-01-01

    Proteo-β-glucan from Maitake (PGM) is a strong immune regulator, and its receptor is called Dectin-1. Cumulative evidence suggests that AMPA receptors are important for the treatment of depression. Here, we report that PGM treatment leads to a significant antidepressant effect in the tail suspension test and forced swim test after sixty minutes of treatment in mice. After five consecutive days of PGM treatment, this antidepressant effect remained. PGM treatment did not show a hyperactive effect in the open field test. PGM significantly enhanced the expression of its receptor Dectin-1, as well as p-GluA1(S845) and GluA1, but not GluA2 or GluA3 in the prefrontal cortex (PFC) after five days of treatment. The Dectin-1 inhibitor Laminarin was able to block the antidepressant effect of PGM. At the synapses of PFC, PGM treatment significantly up-regulated the p-GluA1(S845), GluA1, GluA2, and GluA3 levels. Moreover, PGM’s antidepressant effects and the increase of p-GluA1(S845)/GluA1 lasted for 3 days after stopping treatment. The AMPA-specific antagonist GYKI 52466 was able to block the antidepressant effect of PGM. This study identified PGM as a novel antidepressant with clinical potential and a new antidepressant mechanism for regulating prefrontal Dectin-1/AMPA receptor signalling. PMID:27329257

  17. The role of Dectin-1 in the host defence against fungal infections.

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    Drummond, Rebecca A; Brown, Gordon D

    2011-08-01

    Dectin-1 is an innate immune pattern recognition receptor (PRR) that, through its ability to bind β-glucans, is involved in the recognition of several pathogenic fungi. Dectin-1 can stimulate a variety of cellular responses via the Syk/CARD9 signalling pathway, including phagocytosis, cytokine production and the respiratory burst. Several advances in our understanding of Dectin-1 immunobiology have been made in recent years, including characterisation of additional signalling pathways and demonstration of its ability to directly induce the development of adaptive immunity. However, the physiological role of many of the functions of this receptor is still unclear. This review aims to provide an update on Dectin-1 and its role within antifungal immune responses, focussing on progress made in the last two years.

  18. Concurrent exposure to a dectin-1 agonist suppresses the Th2 response to epicutaneously introduced antigen in mice

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    Lin Jing-Yi

    2013-01-01

    Full Text Available Abstract Background Epicutaneous sensitization with protein allergen that induces predominant Th2 responses is an important sensitization route in atopic dermatitis. Fungal components have been shown to modulate Th cell differentiation. However, the effects of fungal components on epicutaneous sensitization are unclear. Results In this study, we showed that co-administration of curdlan, a dectin-1 agonist, during epicutaneous ovalbumin sensitization of BALB/c mice decreased the IL-5 and IL-13 levels in supernatants of lymph node cell ovalbumin reactivation cultures. Mechanistically, curdlan co-administration decreased IL-4 and IL-1β expressions in draining lymph nodes. Curdlan co-administration also lower the migration of langerin+ CD103- epidermal Langerhans cells into draining lymph nodes at 96 hours post-sensitization which might be attributed to decreased expressions of IL-18 and IL-1β in patched skin. Moreover, adoptive transfer of CFSE-labeled transgenic CD4 T cells confirmed that curdlan co-administration decreased the proliferation and IL-4-production of ovalbumin -specific T cells primed by epidermal Langerhans cells. Conclusions These results indicated that concurrent exposure to a dectin-1 agonist suppresses the epicutaneously induced Th2 response by modulating the cytokine expression profiles in draining LNs and the migration of epidermal Langerhans cells. These results highlight the effects of fungal components on epicutaneous allergen sensitization in atopic diseases.

  19. Oxidative burst and nitric oxide responses in carp macrophages induced by zymosan, MacroGard® and selective dectin-1 agonists suggest recognition by multiple pattern recognition receptors

    DEFF Research Database (Denmark)

    Pietretti, D.; Jiménez, Natalia Ivonne Vera; Hoole, D.;

    2013-01-01

    β-Glucans are glucose polymers that are found in the cell walls of plants, bacteria, certain fungi, mushrooms and the cell wall of baker's yeast. In mammals, myeloid cells express several receptors capable of recognizing β-glucans, with the C-type lectin receptor dectin-1 in conjunction with Toll...... of insoluble β-glucan and mannan, and MacroGard®, a β-glucan-based feed ingredient for farmed animals including several fish species. Both preparations were confirmed TLR2 ligands by measuring activation of HEK293 cells transfected with human TLR2 and CD14, co-transfected with a secreted embryonic alkaline...... phosphatase (SEAP) reporter gene. In addition, dectin-1-specific ligands in mammals i.e. zymosan treated to deplete the TLR-stimulating properties and curdlan, were monitored for their effects on carp macrophages by measuring reactive oxygen and nitrogen radicals production, as well as cytokine gene...

  20. Murine pattern recognition receptor dectin-1 is essential in the development of experimental autoimmune uveoretinitis.

    Science.gov (United States)

    Stoppelkamp, Sandra; Reid, Delyth M; Yeoh, Joyce; Taylor, Julie; McKenzie, Emma J; Brown, Gordon D; Gordon, Siamon; Forrester, John V; Wong, Simon Y C

    2015-10-01

    Mycobacteria in complete Freund's adjuvant (CFA) are an essential component of immunization protocols in a number of autoimmune disease animal models including experimental autoimmune encephalomyelitis and uveoretinitis (EAE and EAU, respectively). We determined the role in EAU of two C-type lectin receptors on myeloid cells that recognize and respond to mycobacteria. Using receptor-specific antibodies and knockout mice, we demonstrated for the first time that the macrophage mannose receptor delays disease development but does not affect severity. In contrast, dectin-1 is critically involved in the development of CFA-mediated EAU. Disease severity is reduced in dectin-1 knockout mice and antibody blockade of dectin-1 during the induction, but not the effector phase, prevents EAU development. Significantly, similar blockade of dectin-1 in vivo has no effect in non-CFA-mediated, spontaneously induced or adoptive transfer models of EAU. Thus dectin-1 plays a critical role in the ability of complete Freund's adjuvant to induce EAU in mice.

  1. Taenia taeniaeformis in rat favors protracted skin lesions caused by Sporothrix schenckii infection: Dectin-1 and IL-17 are dispensable for clearance of this fungus.

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    Xiaohui Zhang

    Full Text Available We occasionally found that cestode Taenia taeniaeformis in rats favored Sporothrix schenckii infection and survival, causing protracted cutaneous lesions. In this study, we compared the pathology and cytokines profile of rats co-infected with the two pathogens and infected with S. schenckii alone to explore underlying mechanisms. In the co-infection group, there was high expression of β-glucan receptor Dectin-1 in the cutaneous lesions and no multinucleated giant cells, but in the S. schenckii infection group the opposite was observed. Cytokines profiles demonstrated an expected finding that IL-4, commonly expressed in helminth and fungus infection, is undetectable in the two infection groups. In the single fungal infection group, cytokines IFN-γ, IL-10 and IL-17 kept increasing in the first few weeks of infection to a peak which was followed by gradual decrease. This study showed that Dectin-1 and IL-17, which were believed to be the major anti-fungus mechanisms, are Th2 independent and dispensable for clearance of S. schenckii infection, suggesting that S. schenckii has a different molecular recognition pattern and evokes anti-infection mechanisms other than Dectin-1 and IL-17.

  2. Taenia taeniaeformis in rat favors protracted skin lesions caused by Sporothrix schenckii infection: Dectin-1 and IL-17 are dispensable for clearance of this fungus.

    Science.gov (United States)

    Zhang, Xiaohui; Zhang, Jing; Huang, Huaiqiu; Xue, Ruzeng; Hu, Xuchu; Li, Meirong; Zhong, Yi; Yuan, Liyan

    2012-01-01

    We occasionally found that cestode Taenia taeniaeformis in rats favored Sporothrix schenckii infection and survival, causing protracted cutaneous lesions. In this study, we compared the pathology and cytokines profile of rats co-infected with the two pathogens and infected with S. schenckii alone to explore underlying mechanisms. In the co-infection group, there was high expression of β-glucan receptor Dectin-1 in the cutaneous lesions and no multinucleated giant cells, but in the S. schenckii infection group the opposite was observed. Cytokines profiles demonstrated an expected finding that IL-4, commonly expressed in helminth and fungus infection, is undetectable in the two infection groups. In the single fungal infection group, cytokines IFN-γ, IL-10 and IL-17 kept increasing in the first few weeks of infection to a peak which was followed by gradual decrease. This study showed that Dectin-1 and IL-17, which were believed to be the major anti-fungus mechanisms, are Th2 independent and dispensable for clearance of S. schenckii infection, suggesting that S. schenckii has a different molecular recognition pattern and evokes anti-infection mechanisms other than Dectin-1 and IL-17.

  3. Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

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    Elena Gabrielli

    Full Text Available We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc of human IgG(1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

  4. Human dendritic cells activated via dectin-1 are efficient at priming Th17, cytotoxic CD8 T and B cell responses.

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    Sudhanshu Agrawal

    Full Text Available BACKGROUND: Dendritic cells capture antigens through PRRs and modulate adaptive immune responses. The type of adaptive immune T cell response generated is dependent upon the type of PRR activated by the microbes. Dectin-1 is a C-type lectin receptor present on dendritic cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that selective dectin-1 agonist Curdlan can activate human DCs and induce the secretion of large amounts of IL-23, IL-1β, IL-6 and low levels of IL-12p70 as determined by ELISA. The Curdlan-stimulated DCs are efficient at priming naïve CD4 cells to differentiate into Th17 and Th1 cells. Furthermore, these CD4 T cells induce differentiation of B cells to secrete IgG and IgA. In addition, Curdlan-stimulated DCs promote the expansion and differentiation of Granzyme and perforin expressing cytotoxic T lymphocyte that display high cytolytic activity against target tumor cells in vitro. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that DCs stimulated through Dectin-1 can generate efficient Th, CTL and B cell responses and can therefore be used as effective mucosal and systemic adjuvants in humans.

  5. Systems biology of host-mycobiota interactions: dissecting Dectin-1 and Dectin-2 signalling in immune cells with DC-ATLAS.

    Science.gov (United States)

    Rizzetto, Lisa; De Filippo, Carlotta; Rivero, Damariz; Riccadonna, Samantha; Beltrame, Luca; Cavalieri, Duccio

    2013-11-01

    Modelling the networks sustaining the fruitful coexistence between fungi and their mammalian hosts is becoming increasingly important to control emerging fungal pathogens. The C-type lectins Dectin-1 and Dectin-2 are involved in host defense mechanisms against fungal infection driving inflammatory and adaptive immune responses and complement in containing fungal burdens. Recognizing carbohydrate structures in pathogens, their engagement induces maturation of dendritic cells (DCs) into potent immuno-stimulatory cells endowed with the capacity to efficiently prime T cells. Owing to these properties, Dectin-1 and Dectin-2 agonists are currently under investigation as promising adjuvants in vaccination procedures for the treatment of fungal infection. Thus, a detailed understanding of events' cascade specifically triggered in DCs upon engagement is of great interest in translational research. Here, we summarize the current knowledge on Dectin-1 and Dectin-2 signalling in DCs highlighting similarities and differences. Detailed maps are annotated, using the Biological Connection Markup Language (BCML) data model, and stored in DC-ATLAS, a versatile resource for the interpretation of high-throughput data generated perturbing the signalling network of DCs. Copyright © 2013 Elsevier GmbH. All rights reserved.

  6. Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages.

    NARCIS (Netherlands)

    Ferwerda, G.; Meyer-Wentrup, F.; Kullberg, B.J.; Netea, M.G.; Adema, G.J.

    2008-01-01

    The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized

  7. Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages

    NARCIS (Netherlands)

    Strijbis, Karin; Tafesse, Fikadu G.; Fairn, Gregory D.; Witte, Martin D.; Dougan, Stephanie K.; Watson, Nicki; Spooner, Eric; Esteban, Alexandre; Vyas, Valmik K.; Fink, Gerald R.; Grinstein, Sergio; Ploegh, Hidde L.

    Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine

  8. Insoluble β-glucan from the cell wall of Candida albicans induces immune responses of human THP-1 monocytes through Dectin-1

    Institute of Scientific and Technical Information of China (English)

    LI Min; LIU Ze-hu; CHEN Qing; ZHOU Wu-qing; YU Mei-wen; L(U) Gui-xia; L(U) Xue-lian; SHEN Yong-nian; LIU Wei-da; WU Shao-xi

    2009-01-01

    Background β-glucan is the major structure component of Candida albicans (C. albicans) cell wall. It has been demonstrated that Dectin-1 as the principal C-type lectin pattern-recognition receptor (PRR) can recognize fungal β-glucan and induce immune responses. In this study, we sought to clarify whether insoluble β-glucan from the cell wall of C. albicans (CalG) could induce immune responses in human THP-1 monocytes (a human acute monocytic leukemia cell line) and to determine the underlying mechanisms.Methods Human THP-1 monocytes were challenged with CalG in vitro. The mRNA expression of Dectin-1, Toll-like receptors (TLR2), proinflammatory cytokine (TNF-α) and chemokine (IL-8) was assayed by real-time reverse transcription polymerase chain reaction (RT-PCR). The secretion of TNF-α and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA). H2O2 release was determined by microplate fluorescent assay. Western blotting was used to analyze IkB-α phosphorylation and degradation.Results Exposure of THP-1 monocytes to CalG led to increased gene expression and secretion of TNF-α and IL-8.CalG induced H2O2 release in a time-dependent manner. CalG hydrolyzed with zymolyase failed to induce gene expression and secretion of TNF-α, IL-8 and H2O2 release. CalG up-regulated the mRNA of Dectin-1, whereas the mRNA level of TLR2 was not altered. THP-1 monocytes challenged with CalG resulted in the activation of NF-KB in a time-dependent manner. Dectin-1 inhibitor laminarin blocked the CalG-induced production of TNF-α and H2O2 in THP-1 monocytes, but no such effect was observed in pretreatment with anti-TLR2 neutralizing antibody and the LPS inhibitor (polymyxin B).Conclusion CalG may play a role in activation of immune responses in human THP-1 cells through Dectin-1, not TLR2.This work was supported by a grant from National Natural ScienceFoundation of China (No. 30671893).

  9. Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

    Institute of Scientific and Technical Information of China (English)

    Xu-Dong; Peng; Gui-Qiu; Zhao; Jing; Lin; Nan; Jiang; Qiang; Xu; Cheng-Cheng; Zhu; Jian-Qiu; Qu; Lin; Cong; Hui; Li

    2015-01-01

    AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

  10. Phospholipase Cgamma2 is critical for Dectin-1-mediated Ca2+ flux and cytokine production in dendritic cells.

    Science.gov (United States)

    Xu, Shengli; Huo, Jianxin; Lee, Koon-Guan; Kurosaki, Tomohiro; Lam, Kong-Peng

    2009-03-13

    Dectin-1 is a C-type lectin that recognizes beta-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-kappaB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca2+ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) gamma-inhibitor U73122, suggesting that Dectin-1 signals via a PLCgamma pathway to induce Ca2+ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCgamma1 and PLCgamma2. However, we show that PLCgamma2 is the critical enzyme for Dectin-1 signaling in DCs. PLCgamma2-deficient DCs have drastic impairment of Ca2+ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor alpha. PLCgamma2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCgamma2-deficient DCs are also impaired in their activation of NF-kappaB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKalpha/beta activation and IkappaBalpha degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca2+ signaling and that PLCgamma2 is a critical player in the Dectin-1 signal transduction pathway.

  11. Dectin-1 Polymorphism: A Genetic Disease Specifier in Autism Spectrum Disorders?

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    Meriem Bennabi

    Full Text Available In autism spectrum disorders (ASD, complex gene-environment interactions contribute to disease onset and progress. Given that gastro-intestinal dysfunctions are common in ASD, we postulated involvement of microbial dysbiosis in ASD and investigated, under a case-control design, the influence of DNA polymorphisms in the CLEC7A gene that encodes a pivotal fungal sensor, Dectin-1.DNAs from 478 ASD patients and 351 healthy controls (HC were analyzed for the CLEC7A rs16910631G/A and rs2078178 A/G single nucleotide polymorphisms (SNPs. Differences in the distribution of allele, genotype and haplotype by Chi-square testing and nonparametric analysis by Kruskal-Wallis/Mann-Whitney tests, where appropriate, were performed. The free statistical package R.2.13 software was used for the statistical analysis.We found that the CLEC7A rs2078178 G allele and GG genotype were more prevalent in HC as compared to ASD but failed to reach statistical significance for the latter (pc = 0.01, 0.06 respectively. However, after phenotype-based stratification, the CLEC7A rs2078178 G allele and GG genotype were found to be significantly more frequent in the Asperger group as compared to other ASD subsets (pc = 0.02, 0.01, a finding reinforced by haplotype analysis (rs2078178/rs16910631 G-G/G-G (pc = 0.002. Further, intellectual quotient (IQ-based stratification of ASD patients revealed that IQ values increase linearly along the CLEC7A rs2078178 AA, AG and GG genotypes (p = 0.05 and in a recessive manner (GG vs. AA+AG p = 0.02, further confirmed by haplotype distribution (CLEC7A rs2078178-16910631; A-G/A-G, A-G/G-G and G-G/G-G, p = 0.02, G-G/G-G vs. others, p = 0.01.Our data suggest that the genetic diversity of CLEC7A gene influences the ASD phenotype by behaving as a disease specifier and imply that the genetic control of innate immune response could determine the ASD phenotype.

  12. Bruton's Tyrosine Kinase (BTK and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

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    Karin Strijbis

    Full Text Available Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

  13. Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors.

    NARCIS (Netherlands)

    Dennehy, K.M.; Ferwerda, G.; Faro-Trindade, I.; Pyz, E.; Willment, J.A.; Taylor, P.R.; Kerrigan, A.; Tsoni, S.V.; Gordon, S.; Meyer-Wentrup, F.; Adema, G.J.; Kullberg, B.J.; Schweighoffer, E.; Tybulewicz, V.; Mora-Montes, H.M.; Gow, N.A.; Williams, D.L.; Netea, M.G.; Brown, G.D.

    2008-01-01

    Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collabor

  14. Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages

    Science.gov (United States)

    Strijbis, Karin; Tafesse, Fikadu G.; Fairn, Gregory D.; Witte, Martin D.; Dougan, Stephanie K.; Watson, Nicki; Spooner, Eric; Esteban, Alexandre; Vyas, Valmik K.; Fink, Gerald R.; Grinstein, Sergio; Ploegh, Hidde L.

    2013-01-01

    Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans. PMID:23825946

  15. Mast cells phagocyte Candida albicans and produce nitric oxide by mechanisms involving TLR2 and Dectin-1.

    Science.gov (United States)

    Pinke, Karen Henriette; Lima, Heliton Gustavo de; Cunha, Fernando Queiroz; Lara, Vanessa Soares

    2016-02-01

    Candida albicans (C. albicans) is a fungus commonly found in the human mucosa, which may cause superficial and systemic infections, especially in immunosuppression. Until now, the main actors in the defense against this fungus are the epithelial cells, neutrophils, macrophages/monocytes and dendritic cells. However, mast cells are strategically located to play a first line of anti-Candida defense and it has appropriate mechanisms to do it. As with other cells, the recognition of C. albicans occurs meanly via TLR2 and Dectin-1. We assess the TLR2/Dectin-1 involvement in phagocytosis and production of nitric oxide (NO) and reactive oxygen species (ROS) by mast cells challenged with C. albicans. Bone marrow-derived mast cells (MC) from wild type (Wt) or knockout (TLR2-/-) mice C57BL/6 were subjected to in vitro Dectin-1 blockade. After challenged with FITC-labeled C. albicans or zymosan, phagocytosis was analyzed by microscopy. The intracellular production of NO and ROS was measured by DAF-FM diacetate and CellROX Deep/Red Reagent kits. The nitrite formation and hydrogen peroxide release were analyzed by Griess reaction and Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. Wt/MC phagocytose C. albicans with production of intracellular NO, but not ROS. Moreover, increased levels of nitrite were also observed. The absence and/or blockade of TLR2/Dectin-1 caused significant decreased in C. albicans phagocytosis and NO production. Our results showed that mast cells are able to phagocytose and produce NO against C. albicans via TLR2/Dectin-1. Therefore, mast cells could be important during the course of Candida infection and as a therapeutic target.

  16. CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

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    Juin-Hua Huang

    2015-07-01

    Full Text Available Collaboration between heterogeneous pattern recognition receptors (PRRs leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3 and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA, genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

  17. Trichoderma asperelloides Spores Downregulate dectin1/2 and TLR2 Receptors of Mice Macrophages and Decrease Candida parapsilosis Phagocytosis Independent of the M1/M2 Polarization

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    Andréa G. dos Santos

    2017-09-01

    had reduced expression of pattern recognition receptors, such as TLR2, dectin-1 and dectin-2, all involved in the first line of defense against clinically important yeasts. Our data could infer that T. asperelloides spores may confer susceptibility to infection by C. parapsilosis.

  18. Participation of dectin-1 receptor on NETs release against Paracoccidioides brasiliensis: Role on extracellular killing.

    Science.gov (United States)

    Bachiega, Tatiana Fernanda; Dias-Melicio, Luciane Alarcão; Fernandes, Reginaldo Keller; de Almeida Balderramas, Helanderson; Rodrigues, Daniela Ramos; Ximenes, Valdecir Farias; de Campos Soares, Ângela Maria Victoriano

    2016-02-01

    Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse.

  19. Dectin-1-Mediated Pathway Contributes to Fusarium proliferatum-Induced CXCL-8 Release from Human Respiratory Epithelial Cells

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    Chang-Ching Yeh

    2017-03-01

    Full Text Available Fusarium species are causative agents of human respiratory disorders and are distributed widely in our environment. Little is known of their interaction with human respiratory epithelial cells, which may contribute to allergic airway responses. In this study, we report on the release of C–X–C motif chemokine ligand 8 (CXCL-8 from human bronchial epithelial BEAS-2B cells upon stimulation with Fusarium proliferatum extracts. F. proliferatum-induced cytokine release from BEAS-2B cells was determined by cytokine array and CXCL-8 enzyme-linked immunosorbent assay (ELISA kits. Blocking antibodies and signaling pathway inhibitors were employed to delineate cell surface receptors and signaling pathways participating in CXCL-8 release. F. proliferatum extracts induced the release of CXCL-8 in a time-dependent manner. The dectin-1 receptor ligands, curdlan and laminarin, reduced CXCL-8 release. Cells pre-treated with anti-Dectin-1 antibodies (2 µg/mL decreased CXCL-8 release by 24%. Furthermore, F. proliferatum-stimulated CXCL-8 release was reduced by 32%, 53%–81%, 40% and 26% after BEAS-2B cells were pretreated with activation inhibitors of spleen tyrosine kinase (Syk—piceatannol—, mitogen-activated protein kinases (MAPKs—PD98059, U0126, SB202190, SP600125—, phosphatidylinositol-3-kinase (PI3K—LY294002—and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB—BAY117082—, respectively. These results suggest that Dectin-1-mediated activation of the Syk, MAPKs, PI3K and NF-κB signaling pathways contributes to F. proliferatum-stimulated CXCL-8 release from BEAS-2B cells and provides an important basis for developing novel therapeutic strategies in clinical allergy.

  20. TLR-4 Cooperates with Dectin-1 and Mannose Receptor to Expand Th17 and Tc17 Cells Induced by Paracoccidioides brasiliensis Stimulated Dendritic Cells

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    Vera Lucia Garcia Calich

    2015-03-01

    Full Text Available The concomitant use of diverse Pattern Recognition Receptors (PRRs by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the Mannose Receptor (MR are C-type Lectin Receptors (CLRs previously reported to cooperate with Toll Like Receptors (TLRs signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4+ and CD8+ T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1 and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs from C57BL/6 mice. Then, WT, Dectin-1-/-, TLR-2-/- and TLR-4-/- DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4+ and CD8+ T cells. In addition, treatment of WT, TLR-2-/- and TLR-4-/- DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1-/- DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was restricted to Dectin-1, TLR

  1. Evolution of the C-Type Lectin-Like Receptor Genes of the DECTIN-1 Cluster in the NK Gene Complex

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    Susanne Sattler

    2012-01-01

    Full Text Available Pattern recognition receptors are crucial in initiating and shaping innate and adaptive immune responses and often belong to families of structurally and evolutionarily related proteins. The human C-type lectin-like receptors encoded in the DECTIN-1 cluster within the NK gene complex contain prominent receptors with pattern recognition function, such as DECTIN-1 and LOX-1. All members of this cluster share significant homology and are considered to have arisen from subsequent gene duplications. Recent developments in sequencing and the availability of comprehensive sequence data comprising many species showed that the receptors of the DECTIN-1 cluster are not only homologous to each other but also highly conserved between species. Even in Caenorhabditis elegans, genes displaying homology to the mammalian C-type lectin-like receptors have been detected. In this paper, we conduct a comprehensive phylogenetic survey and give an up-to-date overview of the currently available data on the evolutionary emergence of the DECTIN-1 cluster genes.

  2. Progress in the research of Dectin-1 and the non-TLR PRR immunologic recognition%Dectin-1与非Toll样模式识别受体免疫识别的研究进展

    Institute of Scientific and Technical Information of China (English)

    王丽; 曹雪涛

    2007-01-01

    Toll样受体(TLR)是一类天然免疫相关的模式识别受体(PRR),在机体抵御感染中发挥着极为重要的作用.除TLR外,目前还发现了很多其它种类PRR,这些非Toll样PRR在天然免疫中的作用也是不可缺少的,它们既可以与TLR协同作用,也可以单独发挥作用.Dectin-1是近年来颇受关注的一种非Toll样PRR,属于NK细胞受体样C型凝集素,是一种Ⅱ型跨膜受体,通过对真菌的识别参与免疫效应细胞的吞噬及杀伤过程,诱导机体产生一系列的细胞因子及趋化因子,从而参与机体防御真菌病原体的天然免疫反应,但在某些情况下也可能引起自身免疫性疾病的发生.

  3. Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

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    Shi Qun Zhang

    2016-05-01

    Full Text Available The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3-glucan, a crucial pathogen-associated molecular pattern (PAMP of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.

  4. A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice.

    Science.gov (United States)

    Yoshitomi, Hiroyuki; Sakaguchi, Noriko; Kobayashi, Katsuya; Brown, Gordon D; Tagami, Tomoyuki; Sakihama, Toshiko; Hirota, Keiji; Tanaka, Satoshi; Nomura, Takashi; Miki, Ichiro; Gordon, Siamon; Akira, Shizuo; Nakamura, Takashi; Sakaguchi, Shimon

    2005-03-21

    A combination of genetic and environmental factors can cause autoimmune disease in animals. SKG mice, which are genetically prone to develop autoimmune arthritis, fail to develop the disease under a microbially clean condition, despite active thymic production of arthritogenic autoimmune T cells and their persistence in the periphery. However, in the clean environment, a single intraperitoneal injection of zymosan, a crude fungal beta-glucan, or purified beta-glucans such as curdlan and laminarin can trigger severe chronic arthritis in SKG mice, but only transient arthritis in normal mice. Blockade of Dectin-1, a major beta-glucan receptor, can prevent SKG arthritis triggered by beta-glucans, which strongly activate dendritic cells in vitro in a Dectin-1-dependent but Toll-like receptor-independent manner. Furthermore, antibiotic treatment against fungi can prevent SKG arthritis in an arthritis-prone microbial environment. Multiple injections of polyinosinic-polycytidylic acid double-stranded RNA also elicit mild arthritis in SKG mice. Thus, specific microbes, including fungi and viruses, may evoke autoimmune arthritis such as rheumatoid arthritis by stimulating innate immunity in individuals who harbor potentially arthritogenic autoimmune T cells as a result of genetic anomalies or variations.

  5. C型凝集素1受体参与人中性粒细胞体外杀伤白念珠菌活性的实验研究%Dectin-1, a C-type lectin receptor, participates in the killing of Candida albicans by human neutrophils: an experimental study

    Institute of Scientific and Technical Information of China (English)

    陈青; 曾荣; 沈永年; 胡素泉; 李岷; 刘维达

    2013-01-01

    目的 探讨人中性粒细胞能否通过C型凝集素1受体(dectin-1)识别白念珠菌细胞壁不溶性β葡聚糖来介导体外杀伤白念珠菌的活性.方法 100 mg/L β葡聚糖体外作用于中性粒细胞1、6、24 h后,实时荧光定量逆转录PCR分析dectin-1和Toll样受体2 mRNA表达水平.100 mg/Lβ葡聚糖体外分别刺激中性粒细胞15 min、2h、6h,或先用dectin-1抑制剂昆布多糖100 mg/L和50 mg/L预处理30 min后,再予100 mg/L β葡聚糖刺激2h,微量培养板荧光分析法检测中性粒细胞H2O2释放水平.昆布多糖预处理的中性粒细胞与白念珠菌体外共培养后,检测菌落形成单位(CFU).结果 β葡聚糖作用中性粒细胞1、6、24 h后,dectin-1 mRNA水平分别为2.8195±0.1669、5.4859±0.7244、3.6041±0.5372,均明显高于空白对照组(均P< 0.01).β葡聚糖刺激中性粒细胞15 min后H2O2水平为(64.55±15.67) μmol/L,2h时为(290.34±30.56)μmol/L,6h时为(208.54±26.88) μmol/L,与空白对照组(22.05±3.99) μmol/L比较,差异均有统计学意义(均P< 0.01);100 mg/L和50 mg/L昆布多糖预处理组分别为(80.45±22.41) μmol/L和(130.42±44.55) μmol/L,与β葡聚糖刺激组相比,分别降低了73%和45%,差异均有统计学意义(P<0.01).昆布多糖能明显抑制中性粒细胞体外杀伤白念珠菌的活性(均P< 0.01).结论 Dectin-1参与人中性粒细胞分泌H2O2以及对白念珠菌杀灭活性,为过继性粒细胞转输治疗系统性念珠菌感染提供初步依据.%Objective To investigate whether human neutrophils kill Candida albicans through recognition of insoluble β-glucan in cell walls of C.albicans (CalG) by dectin-1,a C-type lectin receptor.Methods Neutrophils were obtained from peripheral blood of healthy human subjects and cultured in vitro.Real-time PCR was carried out to quantify the mRNA expressions of dectin-1 and Toll-like receptor 2 (TLR2) in neutrophils challenged with CaIG of 100 mg/L for 1,6,and 24 hours.A Fluoro

  6. Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

    Science.gov (United States)

    de Turris, Valeria; Teloni, Raffaela; Chiani, Paola; Bromuro, Carla; Mariotti, Sabrina; Pardini, Manuela; Nisini, Roberto; Torosantucci, Antonella; Gagliardi, Maria Cristina

    2015-01-01

    Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

  7. Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

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    Valeria de Turris

    Full Text Available Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

  8. Role of Pattern Recognition Receptors in the Modulation of Antimicrobial Peptide Expression in the Corneal Epithelial Innate Response to F. solani

    Science.gov (United States)

    Kolar, Satya Sree; Baidouri, Hasna; McDermott, Alison M.

    2017-01-01

    Purpose Fusarium solani (F. solani) keratitis is a potentially sight-threatening fungal infection of the cornea. Antimicrobial peptides (AMPs), such as human β-defensins (hBDs) and cathelicidins, essential components of the immune system, likely have a protective role against F. solani keratitis. We examined the role of pattern recognition receptors (PRRs), Dectin-1, and TLR2 in F. solani–induced modulation of AMP expression in vitro. Methods Human corneal epithelial cells (HCECs) were exposed to heat-inactivated F. solani or pathogen-associated molecular patterns (PAMPs) of F. solani (Zymosan or Zymosan Depleted) for 6, 12, or 24 hours following which AMP mRNA and protein levels were determined. Involvement of TLR2 and Dectin-1 was confirmed by using siRNA knock-down (TLR2 and Dectin-1) or chemical inhibitor BAY 61-3606 (Dectin-1). The functional significance of AMP upregulation was tested using culture supernatant from F. solani or PAMP-treated HCECs against F. solani in the presence of hBD2 or LL37 neutralizing antibody. Results We confirm that HCECs express Dectin-1 and TLR2. HCECs demonstrated upregulation of AMPs hBD2 and cathelicidin LL37 following exposure to heat-inactivated F. solani or PAMPs. TLR2 and Dectin-1 knockdown and BAY 61-3606 treatment decreased AMP mRNA upregulation confirming PRR involvement. The culture supernatant from F. solani or PAMP-treated HCECs showed substantial killing of F. solani and hBD2 or LL37 neutralizing antibody significantly decreased this effect implicating involvement of these AMPs. Conclusions These findings demonstrate that Dectin-1 and TLR2 have an important role in regulating F. solani-induced AMP expression in corneal epithelial cells. PMID:28460048

  9. Effects of immunosuppressive agent on mRNA expression of anti-aspergillus infection-associated receptors on alveolar macrophage%免疫抑制剂对小鼠肺泡巨噬细胞抗曲霉感染相关受体mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    荣令; 周新; 何牡丹; 李峰

    2009-01-01

    目的 探讨免疫抑制剂对小鼠肺泡巨噬细胞抗曲霉感染相关受体mRNA表达的影响.方法 30只昆明小鼠随机分为2组:正常对照组(6只)和免疫抑制组(24只,环磷酰胺150 mg/kg腹腔注射).免疫抑制组小鼠注射环磷酰胺后4 h、8 h、16 h及24 h,分别随机取6只进行支气管肺泡灌洗,收集肺泡巨噬细胞.正常对照组小鼠注射生理盐水24 h后处死,收集肺泡巨噬细胞.使用逆转录-聚合酶链反应检测小鼠肺泡巨噬细胞Toll样受体2(TLR2)、TLR4、树突状细胞相关C型凝集素1(Dectine-1)mRNA表达变化.结果 与正常对照组比较,腹腔注射环磷酰胺4 h后,TLR2 mRNA表达即出现显著下降(P<0.01);在腹腔注射环磷酰胺8 h后TLR4 mRNA表达出现显著下降(P<0.01);Dectine-1 mRNA在腹腔注射环磷酰胺后无明显变化.结论 免疫抑制剂环磷酰胺能够下调肺泡巨噬细胞抗曲霉感染相关受体TRL2和TLR4 mRNA的表达,对Dectine-1 mRNA的表达未见明显影响.%Objective To investigate the effects of immunosuppressive agent on mRNA expression of anti-aspergillus infection-associated receptors on alveolar macrophage.Methods Thirty Kunming mice were randomly divided into two groups:normal control group(n=6)and immunocompromise group(150 mg/kg cyclophosphamide,intraperitoneal injection,n=24).Six mice were randomly taken to collect alveolar macrophage by bronchoalveolar lavage from immunocompromise group at time of 4,8,12 and 24 hours after immunosuppression.Mice in normal control group were sacrificed to collect alveolar macrophage after receiving normal saline intraperitoneal injection.Reverse transcription-polymerase chain reaction was used for determining the mRNA expression of Toll-like receptor 2(TLR2),TLR4 and dendritic cell-associated C-type lectin 1(Dectine-1).Results TLR2 mRNA expression decreased signiffieantly four hours after cyclophosphamide intraperitoneal injection,and decreased continually to 24 hours(all P<0.01).The

  10. Cortical control of facial expression.

    Science.gov (United States)

    Müri, René M

    2016-06-01

    The present Review deals with the motor control of facial expressions in humans. Facial expressions are a central part of human communication. Emotional face expressions have a crucial role in human nonverbal behavior, allowing a rapid transfer of information between individuals. Facial expressions can be either voluntarily or emotionally controlled. Recent studies in nonhuman primates and humans have revealed that the motor control of facial expressions has a distributed neural representation. At least five cortical regions on the medial and lateral aspects of each hemisphere are involved: the primary motor cortex, the ventral lateral premotor cortex, the supplementary motor area on the medial wall, and the rostral and caudal cingulate cortex. The results of studies in humans and nonhuman primates suggest that the innervation of the face is bilaterally controlled for the upper part and mainly contralaterally controlled for the lower part. Furthermore, the primary motor cortex, the ventral lateral premotor cortex, and the supplementary motor area are essential for the voluntary control of facial expressions. In contrast, the cingulate cortical areas are important for emotional expression, because they receive input from different structures of the limbic system.

  11. Social control and expressed emotion.

    Science.gov (United States)

    Greenley, J R

    1986-01-01

    Research shows a higher risk of relapse among schizophrenics in high "expressed emotion" families. In this paper, the measure called "expressed emotion" is conceptualized as an indicator of family attempts to socially control the schizophrenic person's behavior in a particular way. This social control conceptualization is supported by a review of the type of information in the measure. Hypotheses following from this view are examined to assess the construct validity of the measure conceptualized as a type of social control. First, attempts at control are hypothesized to be ways anxious and fearful families try to cope. Second, the family's recognition of the schizophrenic's problem as mental illness is hypothesized to reduce the fearful and anxious family's likelihood of an intense interpersonal social control coping response. This type of social control of involuntary illness behaviors would be abandoned as unjust and unlikely to be effective. Data from the pioneering 1972 study by Brown, Birley, and Wing (Br. J. Psychiatry 121:241-258) provide support for these hypotheses and thus provide support for this social control conceptualization.

  12. Translational control of gene expression and disease

    NARCIS (Netherlands)

    Calkhoven, Cornelis F; Müller, Christine; Leutz, Achim

    2002-01-01

    In the past decade, translational control has been shown to be crucial in the regulation of gene expression. Research in this field has progressed rapidly, revealing new control mechanisms and adding constantly to the list of translationally regulated genes. There is accumulating evidence that trans

  13. Sex Roles and Yielded/Expressed Self-Control.

    Science.gov (United States)

    Ganong, Lawrence H.; Coleman, Marilyn

    1987-01-01

    Results of a study of the impact of sex and sex role orientation on reported self-control behaviors showed that sex did not affect self-control or satisfaction with self-control, but sex role orientation did. Androgynous persons reported using more expressed self-control than others. (PS)

  14. Sex Roles and Yielded/Expressed Self-Control.

    Science.gov (United States)

    Ganong, Lawrence H.; Coleman, Marilyn

    1987-01-01

    Results of a study of the impact of sex and sex role orientation on reported self-control behaviors showed that sex did not affect self-control or satisfaction with self-control, but sex role orientation did. Androgynous persons reported using more expressed self-control than others. (PS)

  15. Control of alphavirus-based gene expression using engineered riboswitches.

    Science.gov (United States)

    Bell, Christie L; Yu, Dong; Smolke, Christina D; Geall, Andrew J; Beard, Clayton W; Mason, Peter W

    2015-09-01

    Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust gene expression and immune responses. To further explore their use in vaccination, replicons were engineered to allow conditional control over their gene expression. Riboswitches, comprising a ribozyme actuator and RNA aptamer sensor, were engineered into the replicon 3' UTR. Binding of ligand to aptamer modulates ribozyme activity and, therefore, gene expression. Expression from DNA-launched and VRP-packaged replicons containing riboswitches was successfully regulated, achieving a 47-fold change in expression and modulation of the resulting type I interferon response. Moreover, we developed a novel control architecture where riboswitches were integrated into the 3' and 5' UTR of the subgenomic RNA region of the TC-83 virus, leading to an 1160-fold regulation of viral replication. Our studies demonstrate that the use of riboswitches for control of RNA replicon expression and viral replication holds promise for development of novel and safer vaccination strategies.

  16. Evidence for mitochondrial genetic control of autosomal gene expression.

    Science.gov (United States)

    Kassam, Irfahan; Qi, Tuan; Lloyd-Jones, Luke; Holloway, Alexander; Jan Bonder, Marc; Henders, Anjali K; Martin, Nicholas G; Powell, Joseph E; Franke, Lude; Montgomery, Grant W; Visscher, Peter M; McRae, Allan F

    2016-10-18

    The mitochondrial and nuclear genomes coordinate and co-evolve in eukaryotes in order to adapt to environmental changes. Variation in the mitochondrial genome is capable of affecting expression of genes on the nuclear genome. Sex-specific mitochondrial genetic control of gene expression has been demonstrated in Drosophila melanogaster, where males were found to drive most of the total variation in gene expression. This has potential implications for male-related health and disease resulting from variation in mtDNA solely inherited from the mother. We used a family-based study comprised of 47,323 gene expression probes and 78 mitochondrial SNPs (mtSNPs) from n = 846 individuals to examine the extent of mitochondrial genetic control of gene expression in humans. This identified 15 significant probe-mtSNP associations (P[Formula: see text]) corresponding to 5 unique genes on the mitochondrial and nuclear genomes, with three of these genes corresponding to mitochondrial genetic control of gene expression in the nuclear genome. The associated mtSNPs for three genes (one cis and two trans associations) were replicated (P expression in any of these five probes. Sex-specific effects were examined by applying our analysis to males and females separately and testing for differences in effect size. The MEST gene was identified as having the most significantly different effect sizes across the sexes (P [Formula: see text]). MEST was similarly expressed in males and females with the G allele; however, males with the C allele are highly expressed for MEST, while females show no expression of the gene. This study provides evidence for the mitochondrial genetic control of expression of several genes in humans, with little evidence found for sex-specific effects.

  17. Doing play: competency, control, and expression.

    Science.gov (United States)

    Miller, Stacey; Reid, Denise

    2003-12-01

    This qualitative study investigated the personal experiences of children with cerebral palsy engaging in a virtual reality play intervention program. The study involved in-depth, focused interviews that were conducted with 19 participants aged 8-13 who had a diagnosis of cerebral palsy. A constant comparative inductive method of analysis was used, and several themes emerged. Findings were interpreted using the theory of flow, the theory of self-efficacy, and the model of playfulness in virtual reality computer interactions. The three themes uncovered in the data include (a) doing play, (b) it's my way that matters, and (c) how I see me. The sub-theme safety also emerged. Findings from this study showed that children with physical disabilities are often limited in their play experience compared to their peers without physical disabilities. Children perceived engagement in a virtual reality play intervention program as an enjoyable experience which increased their self-competence and self-efficacy. Participants experienced a sense of control and mastery over the virtual environment and were provided a safe way to explore and challenge their abilities. Participants perceived experiencing flow and reported perceived physical changes and increased social acceptance from both peers and family. These findings provide evidence that virtual reality continues to show promise as a pediatric rehabilitation play intervention tool.

  18. Spatial and temporal control of transgene expression in zebrafish.

    Directory of Open Access Journals (Sweden)

    Alexander A Akerberg

    Full Text Available Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.

  19. Sequential logic model deciphers dynamic transcriptional control of gene expressions.

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    Zhen Xuan Yeo

    Full Text Available BACKGROUND: Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented here. METHODOLOGY: Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM is developed to decipher control mechanisms of dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. PRINCIPAL FINDINGS: SLM is verified by a set of well studied expression data on endo16 of Strongylocentrotus purpuratus (sea urchin during the embryonic midgut development. A dynamic regulatory mechanism for endo16 expression controlled by three binding sites, UI, R and Otx is identified and demonstrated to be consistent with experimental findings. Furthermore, we show that during transition from specification to differentiation in wild type endo16 expression profile, SLM reveals three binary activities are not sufficient to explain the transcriptional regulation of endo16 expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. CONCLUSIONS/SIGNIFICANCE: The sequential logic formalism allows for a simplification of regulation network dynamics going from a continuous to a discrete representation of gene activation in time. In effect our SLM is non-parametric and model-independent, yet

  20. Genetic control of human brain transcript expression in Alzheimer disease.

    Science.gov (United States)

    Webster, Jennifer A; Gibbs, J Raphael; Clarke, Jennifer; Ray, Monika; Zhang, Weixiong; Holmans, Peter; Rohrer, Kristen; Zhao, Alice; Marlowe, Lauren; Kaleem, Mona; McCorquodale, Donald S; Cuello, Cindy; Leung, Doris; Bryden, Leslie; Nath, Priti; Zismann, Victoria L; Joshipura, Keta; Huentelman, Matthew J; Hu-Lince, Diane; Coon, Keith D; Craig, David W; Pearson, John V; Heward, Christopher B; Reiman, Eric M; Stephan, Dietrich; Hardy, John; Myers, Amanda J

    2009-04-01

    We recently surveyed the relationship between the human brain transcriptome and genome in a series of neuropathologically normal postmortem samples. We have now analyzed additional samples with a confirmed pathologic diagnosis of late-onset Alzheimer disease (LOAD; final n = 188 controls, 176 cases). Nine percent of the cortical transcripts that we analyzed had expression profiles correlated with their genotypes in the combined cohort, and approximately 5% of transcripts had SNP-transcript relationships that could distinguish LOAD samples. Two of these transcripts have been previously implicated in LOAD candidate-gene SNP-expression screens. This study shows how the relationship between common inherited genetic variants and brain transcript expression can be used in the study of human brain disorders. We suggest that studying the transcriptome as a quantitative endo-phenotype has greater power for discovering risk SNPs influencing expression than the use of discrete diagnostic categories such as presence or absence of disease.

  1. SinR controls enterotoxin expression in Bacillus thuringiensis biofilms.

    Directory of Open Access Journals (Sweden)

    Annette Fagerlund

    Full Text Available The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism. Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

  2. Genome engineering and gene expression control for bacterial strain development.

    Science.gov (United States)

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen.

  3. Small molecule control of virulence gene expression in Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    James C Charity

    2009-10-01

    Full Text Available In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS, the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp, to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression.

  4. Optogenetic switches for light-controlled gene expression in yeast.

    Science.gov (United States)

    Salinas, Francisco; Rojas, Vicente; Delgado, Verónica; Agosin, Eduardo; Larrondo, Luis F

    2017-04-01

    Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

  5. Controlling gene expression by DNA mechanics: emerging insights and challenges

    OpenAIRE

    Levens, David; Baranello, Laura; Kouzine, Fedor

    2016-01-01

    Transcription initiation is a major control point for the precise regulation of gene expression. Our knowledge of this process has been mainly derived from protein-centric studies wherein cis-regulatory DNA sequences play a passive role, mainly in arranging the protein machinery to coalesce at the transcription start sites of genes in a spatial and temporal-specific manner. However, this is a highly dynamic process in which molecular motors such as RNA polymerase II (RNAPII), helicases, and o...

  6. Differential control of Mincle-dependent cord factor recognition and macrophage responses by the transcription factors C/EBPβ and HIF1α.

    Science.gov (United States)

    Schoenen, Hanne; Huber, Alexandra; Sonda, Nada; Zimmermann, Stephanie; Jantsch, Jonathan; Lepenies, Bernd; Bronte, Vincenzo; Lang, Roland

    2014-10-01

    Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD

  7. Regions of KCNQ K+ Channels Controlling Functional Expression

    Directory of Open Access Journals (Sweden)

    Frank eChoveau

    2012-10-01

    Full Text Available KCNQ1-5 α-subunits assemble to form K+ channels that play critical roles in the function of numerous tissues. The channels are tetramers of subunits containing six transmembrane domains. Each subunit consists of a pore region (S5-pore-S6 and a voltage sensor domain (S1-S4. Despite similar structures, KCNQ2 and KCNQ3 homomers yield small current amplitudes compared to other KCNQ homomers and KCNQ2/3 heteromers. Two major mechanisms have been suggested as governing functional expression. The first involves control of channel trafficking to the plasma membrane by the distal part of the C-terminus, containing two coiled-coiled domains, required for channel trafficking and assembly. The proximal half of the C-terminus is the crucial region for channel modulation by signaling molecules such as calmodulin, which may mediate C- and N-terminal interactions. The N-terminus of KCNQ channels has also been postulated as critical for channel surface expression. The second mechanism suggests networks of interactions between the pore helix and the selectivity filter, and between the pore helix and the S6 domain that govern KCNQ current amplitudes. Here, we summarize the role of these different regions in expression of functional KCNQ channels.

  8. Control levels of acetylcholinesterase expression in the mammalian skeletal muscle.

    Science.gov (United States)

    Grubic, Z; Zajc-Kreft, K; Brank, M; Mars, T; Komel, R; Miranda, A F

    1999-05-14

    Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early

  9. [Control of growth and expression of protooncogenes in regenerating liver].

    Science.gov (United States)

    Zou, Y; Gong, D Z; Cui, X Y; Mei, M H

    1996-01-01

    There are many humoral factors involved in the control of growth in regenerating liver. The complete hepatocyte mitogens such as hepatocyte growth factor (HGF), hepatic stimulator substance (HSS) can strongly stimulate hepatocyte DNA synthesis and mitosis. The hepatocyte growth inhibitors such as transforming growth factor beta 1 (TGF beta 1), however, do not stimulate DNA synthesis, but inhibit EGF mitogenesis. In addition, the comitogens such as norepinephrine and insulin are necessary to regulate the growth of regenerating liver. It has become clear that the hepatocyte proliferation and protooncogenes are linked closely. Some protooncogenes can express specifically as markers in the different phases of the cell cycle and in hepatocytes that enter the cell cycle (G0 to G1 transit) and continue to progress.

  10. Controlling gene expression by DNA mechanics: emerging insights and challenges.

    Science.gov (United States)

    Levens, David; Baranello, Laura; Kouzine, Fedor

    2016-11-01

    Transcription initiation is a major control point for the precise regulation of gene expression. Our knowledge of this process has been mainly derived from protein-centric studies wherein cis-regulatory DNA sequences play a passive role, mainly in arranging the protein machinery to coalesce at the transcription start sites of genes in a spatial and temporal-specific manner. However, this is a highly dynamic process in which molecular motors such as RNA polymerase II (RNAPII), helicases, and other transcription factors, alter the level of mechanical force in DNA, rather than simply a set of static DNA-protein interactions. The double helix is a fiber that responds to flexural and torsional stress, which if accumulated, can affect promoter output as well as change DNA and chromatin structure. The relationship between DNA mechanics and the control of early transcription initiation events has been under-investigated. Genomic techniques to display topological stress and conformational variation in DNA across the mammalian genome provide an exciting new insight on the role of DNA mechanics in the early stages of the transcription cycle. Without understanding how torsional and flexural stresses are generated, transmitted, and dissipated, no model of transcription will be complete and accurate.

  11. An autonomous molecular computer for logical control of gene expression

    Science.gov (United States)

    Benenson, Yaakov; Gil, Binyamin; Ben-Dor, Uri; Adar, Rivka; Shapiro, Ehud

    2004-05-01

    Early biomolecular computer research focused on laboratory-scale, human-operated computers for complex computational problems. Recently, simple molecular-scale autonomous programmable computers were demonstrated allowing both input and output information to be in molecular form. Such computers, using biological molecules as input data and biologically active molecules as outputs, could produce a system for `logical' control of biological processes. Here we describe an autonomous biomolecular computer that, at least in vitro, logically analyses the levels of messenger RNA species, and in response produces a molecule capable of affecting levels of gene expression. The computer operates at a concentration of close to a trillion computers per microlitre and consists of three programmable modules: a computation module, that is, a stochastic molecular automaton; an input module, by which specific mRNA levels or point mutations regulate software molecule concentrations, and hence automaton transition probabilities; and an output module, capable of controlled release of a short single-stranded DNA molecule. This approach might be applied in vivo to biochemical sensing, genetic engineering and even medical diagnosis and treatment. As a proof of principle we programmed the computer to identify and analyse mRNA of disease-related genes associated with models of small-cell lung cancer and prostate cancer, and to produce a single-stranded DNA molecule modelled after an anticancer drug.

  12. Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2010-02-01

    Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

  13. Expressing emotions through vibration for perception and control

    OpenAIRE

    Ur Réhman, Shafiq

    2010-01-01

    This thesis addresses a challenging problem: “how to let the visually impaired ‘see’ others emotions”. We, human beings, are heavily dependent on facial expressions to express ourselves. A smile shows that the person you are talking to is pleased, amused, relieved etc. People use emotional information from facial expressions to switch between conversation topics and to determine attitudes of individuals. Missing emotional information from facial expressions and head gestures makes the visuall...

  14. AMO EXPRESS: A Command and Control Experiment for Crew Autonomy

    Science.gov (United States)

    Stetson, Howard K.; Frank, Jeremy; Cornelius, Randy; Haddock, Angie; Wang, Lui; Garner, Larry

    2015-01-01

    NASA is investigating a range of future human spaceflight missions, including both Mars-distance and Near Earth Object (NEO) targets. Of significant importance for these missions is the balance between crew autonomy and vehicle automation. As distance from Earth results in increasing communication delays, future crews need both the capability and authority to independently make decisions. However, small crews cannot take on all functions performed by ground today, and so vehicles must be more automated to reduce the crew workload for such missions. NASA's Advanced Exploration Systems Program funded Autonomous Mission Operations (AMO) project conducted an autonomous command and control demonstration of intelligent procedures to automatically initialize a rack onboard the International Space Station (ISS) with power and thermal interfaces, and involving core and payload command and telemetry processing, without support from ground controllers. This autonomous operations capability is enabling in scenarios such as a crew medical emergency, and representative of other spacecraft autonomy challenges. The experiment was conducted using the Expedite the Processing of Experiments for Space Station (EXPRESS) rack 7, which was located in the Port 2 location within the U.S Laboratory onboard the International Space Station (ISS). Activation and deactivation of this facility is time consuming and operationally intensive, requiring coordination of three flight control positions, 47 nominal steps, 57 commands, 276 telemetry checks, and coordination of multiple ISS systems (both core and payload). The autonomous operations concept includes a reduction of the amount of data a crew operator is required to verify during activation or de-activation, as well as integration of procedure execution status and relevant data in a single integrated display. During execution, the auto-procedures provide a step-by-step messaging paradigm and a high level status upon termination. This

  15. Multiple Signaling Pathways Control Tbx6 Expression during Xenopus Myogenesis

    Institute of Scientific and Technical Information of China (English)

    Pan-Feng FANG; Rui-Ying HU; Xing-Yue HE; Xiao-Yan DING

    2004-01-01

    Tbx6 is critical for somite specification and myogenesis initiation.It has been shown that Activin/Nodal,VegT/Nodal,FGF,and BMP signaling pathways are involved early in specifying mesoderm or later in patterning mesoderm,and Xnot plays roles in setting up the boundary between notochord and paraxial mesoderm.In this study,we introduce the dominant negative form of above genes into embryos to evaluate if they are responsible for regulating Tbx6 expression.The results show that: (1)Activin/Nodal and VegT/Nodal signals are necessary for both initiation and maintenance of Tbx6 expression,and Nodal is sufficient to induce ectopic Tbx6 expression;(2) FGF signal is necessary for the initiation and maintenance of Tbx6,but it is not sufficient to induce Tbx6 expression;(3) BMP is also necessary for the expression of Tbx6,and the induction of Tbx6 expression by BMP is dose dependent;(4) Xnot has no effect on the expression of Tbx6.Our results suggest that several signaling pathways are involved in regulating Tbx6expression,and pave the route to reveal the molecular mechanism of initiating myogenesis.

  16. The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting.

    Science.gov (United States)

    Köhler, Claudia; Page, Damian R; Gagliardini, Valeria; Grossniklaus, Ueli

    2005-01-01

    The maternally expressed Arabidopsis thaliana Polycomb group protein MEDEA (MEA) controls expression of the MADS-box gene PHERES1 (PHE1). Here, we show that PHE1 is mainly paternally expressed but maternally repressed and that this maternal repression of PHE1 breaks down in seeds lacking maternal MEA activity. Because Polycomb group proteins control parental imprinting in mammals as well, the independent recruitment of similar protein machineries for the imprinting of genes is a notable example of convergent evolution.

  17. Contextual control over expression of fear is affected by cortisol

    Directory of Open Access Journals (Sweden)

    Vanessa Anna Van Ast

    2012-10-01

    Full Text Available At the core of anxiety disorders is the inability to use contextual information to modulate behavioral responses to potentially threatening events. Models of the pathogenesis of anxiety disorders incorporate stress and concomitant stress hormones as important vulnerability factors, while others emphasize sex as an important factor. However, translational basic research has not yet investigated the effects of stress hormones and sex on the ability to use contextual information to modulate responses to threat. Therefore, the purpose of the present study was threefold: first, we aimed at developing an experimental paradigm specifically capable of capturing contextual modulation of the expression of fear. Second, we tested whether cortisol would alter the contextualization of fear expression. Third, we aimed at assessing whether alterations in contextualization due to cortisol were different for men and women. Healthy participants (n = 42 received placebo or hydrocortisone (20 mg prior to undergoing a newly developed differential contextual fear conditioning paradigm. The results indicated that people rapidly acquire differential contextual modulation of the expression of fear, as measured by fear potentiated startle and skin conductance responses. In addition, cortisol impaired the contextualization of fear expression leading to increased fear generalization on fear potentiated startle data in women. The opposite pattern was found in men. Finally, as assessed by skin conductance responses, cortisol impaired differential conditioning in men. The results are in line with models suggesting heightened vulnerability in women for developing anxiety disorders after stressful events.

  18. A modified consumer inkjet for spatiotemporal control of gene expression.

    Directory of Open Access Journals (Sweden)

    Daniel J Cohen

    Full Text Available This paper presents a low-cost inkjet dosing system capable of continuous, two-dimensional spatiotemporal regulation of gene expression via delivery of diffusible regulators to a custom-mounted gel culture of E. coli. A consumer-grade, inkjet printer was adapted for chemical printing; E. coli cultures were grown on 750 microm thick agar embedded in micro-wells machined into commercial compact discs. Spatio-temporal regulation of the lac operon was demonstrated via the printing of patterns of lactose and glucose directly into the cultures; X-Gal blue patterns were used for visual feedback. We demonstrate how the bistable nature of the lac operon's feedback, when perturbed by patterning lactose (inducer and glucose (inhibitor, can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this include sharp boundaries and the generation of traveling waves of mRNA expression. To our knowledge, this is the first demonstration of reaction-diffusion effects in the well-studied lac operon. A finite element reaction-diffusion model of the lac operon is also presented which predicts pattern formation with good fidelity.

  19. Control of gene expression by CRISPR-Cas systems

    Science.gov (United States)

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems. PMID:24273648

  20. Simple Expressions for Safety Factors in Inventory Control

    NARCIS (Netherlands)

    Strijbosch, L.W.G.; Moors, J.J.A.

    1999-01-01

    The literature on inventory control discusses many methods to establish the level of decision parameters -like reorder levels or safety factors-, necessary to attain a prescribed service level. In general, however, these methods are not easy applicable: they often use time-consuming iterations, requ

  1. Parvalbumin-expressing interneurons linearly control olfactory bulb output.

    Science.gov (United States)

    Kato, Hiroyuki K; Gillet, Shea N; Peters, Andrew J; Isaacson, Jeffry S; Komiyama, Takaki

    2013-12-04

    In the olfactory bulb, odor representations by principal mitral cells are modulated by local inhibitory circuits. While dendrodendritic synapses between mitral and granule cells are typically thought to be a major source of this modulation, the contributions of other inhibitory neurons remain unclear. Here we demonstrate the functional properties of olfactory bulb parvalbumin-expressing interneurons (PV cells) and identify their important role in odor coding. Using paired recordings, we find that PV cells form reciprocal connections with the majority of nearby mitral cells, in contrast to the sparse connectivity between mitral and granule cells. In vivo calcium imaging in awake mice reveals that PV cells are broadly tuned to odors. Furthermore, selective PV cell inactivation enhances mitral cell responses in a linear fashion while maintaining mitral cell odor preferences. Thus, dense connections between mitral and PV cells underlie an inhibitory circuit poised to modulate the gain of olfactory bulb output.

  2. Light Controlled Modulation of Gene Expression by Chemical Optoepigenetic Probes

    Science.gov (United States)

    Reis, Surya A.; Ghosh, Balaram; Hendricks, J. Adam; Szantai-Kis, D. Miklos; Törk, Lisa; Ross, Kenneth N.; Lamb, Justin; Read-Button, Willis; Zheng, Baixue; Wang, Hongtao; Salthouse, Christopher; Haggarty, Stephen J.; Mazitschek, Ralph

    2016-01-01

    Epigenetic gene regulation is a dynamic process orchestrated by chromatin-modifying enzymes. Many of these master regulators exert their function through covalent modification of DNA and histone proteins. Aberrant epigenetic processes have been implicated in the pathophysiology of multiple human diseases. Small-molecule inhibitors have been essential to advancing our understanding of the underlying molecular mechanisms of epigenetic processes. However, the resolution offered by small molecules is often insufficient to manipulate epigenetic processes with high spatio-temporal control. Here, we present a novel and generalizable approach, referred to as ‘Chemo-Optical Modulation of Epigenetically-regulated Transcription’ (COMET), enabling high-resolution, optical control of epigenetic mechanisms based on photochromic inhibitors of human histone deacetylases using visible light. COMET probes may translate into novel therapeutic strategies for diseases where conditional and selective epigenome modulation is required. PMID:26974814

  3. Neural correlates of conflict control on facial expressions with a flanker paradigm.

    Science.gov (United States)

    Liu, Tongran; Xiao, Tong; Shi, Jian-Nong

    2013-01-01

    Conflict control is an important cognitive control ability and it is also crucial for human beings to execute conflict control on affective information. To address the neural correlates of cognitive control on affective conflicts, the present study recorded event-related potentials (ERPs) during a revised Eriksen Flanker Task. Participants were required to indicate the valence of the central target expression while ignoring the flanker expressions in the affective congruent condition, affective incongruent condition and neutral condition (target expressions flanked by scramble blocks). Behavioral results manifested that participants exhibited faster response speed in identifying neutral target face when it was flanked by neutral distractors than by happy distractors. Electrophysiological results showed that happy target expression induced larger N2 amplitude when flanked by sad distractors than by happy distractors and scramble blocks during the conflict monitoring processing. During the attentional control processing, happy target expression induced faster P3 response when it was flanked by happy distractors than by sad distractors, and sad target expression evoked larger P3 amplitude when it was flanked by happy distractors comparing with sad distractors. Taken together, the current findings of temporal dynamic of brain activity during cognitive control on affective conflicts shed light on the essential relationship between cognitive control and affective information processing.

  4. Neural correlates of conflict control on facial expressions with a flanker paradigm.

    Directory of Open Access Journals (Sweden)

    Tongran Liu

    Full Text Available Conflict control is an important cognitive control ability and it is also crucial for human beings to execute conflict control on affective information. To address the neural correlates of cognitive control on affective conflicts, the present study recorded event-related potentials (ERPs during a revised Eriksen Flanker Task. Participants were required to indicate the valence of the central target expression while ignoring the flanker expressions in the affective congruent condition, affective incongruent condition and neutral condition (target expressions flanked by scramble blocks. Behavioral results manifested that participants exhibited faster response speed in identifying neutral target face when it was flanked by neutral distractors than by happy distractors. Electrophysiological results showed that happy target expression induced larger N2 amplitude when flanked by sad distractors than by happy distractors and scramble blocks during the conflict monitoring processing. During the attentional control processing, happy target expression induced faster P3 response when it was flanked by happy distractors than by sad distractors, and sad target expression evoked larger P3 amplitude when it was flanked by happy distractors comparing with sad distractors. Taken together, the current findings of temporal dynamic of brain activity during cognitive control on affective conflicts shed light on the essential relationship between cognitive control and affective information processing.

  5. Shared control of gene expression in bacteria by transcription factors and global physiology of the cell.

    NARCIS (Netherlands)

    Berthoumieux, S.; Jong, H. de; Baptist, G.; Pinel, C.; Ranquet, C.; Ropers, D.; Geiselmann, J.

    2013-01-01

    Gene expression is controlled by the joint effect of (i) the global physiological state of the cell, in particular the activity of the gene expression machinery, and (ii) DNA-binding transcription factors and other specific regulators. We present a model-based approach to distinguish between these t

  6. Facial and Bodily Expressions for Control and Adaptation of Games (ECAG 2008)

    NARCIS (Netherlands)

    Nijholt, Antinus; Poppe, Ronald Walter; Unknown, [Unknown

    2008-01-01

    In this workshop of the 8th IEEE International Conference on Automatic Face and Gesture Recognition (FG 2008), the emphasis is on research on facial and bodily expressions for the control and adaptation of games. We distinguish between two forms of expressions, depending on whether the user has the

  7. AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000

    Science.gov (United States)

    Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and is active while the bacteria are associa...

  8. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  9. Dectin-1 interaction with tetraspanin CD37 inhibits IL-6 production.

    NARCIS (Netherlands)

    Meyer-Wentrup, F.A.G.; Figdor, C.G.; Ansems, M.; Brossart, P.; Wright, M.D.; Adema, G.J.; Spriel, A.B. van

    2007-01-01

    C-type lectins are pattern-recognition receptors important for pathogen binding and uptake by APCs. Evidence is accumulating that integration of incoming cellular signals in APCs is regulated by grouping of receptors and signaling molecules into organized membrane complexes, such as lipid rafts and

  10. Control of cystic fibrosis transmembrane conductance regulator expression by BAP31.

    Science.gov (United States)

    Lambert, G; Becker, B; Schreiber, R; Boucherot, A; Reth, M; Kunzelmann, K

    2001-06-08

    Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is stringently controlled by molecular chaperones participating in formation of the quality control system. It has been shown that about 75% of all CFTR protein and close to 100% of the [DeltaPhe(508)] CFTR variant are rapidly degraded before leaving the endoplasmic reticulum (ER). B cell antigen receptor-associated proteins (BAPs) are ubiquitously expressed integral membrane proteins that may control association with the cytoskeleton, vesicular transport, or retrograde transport from the cis Golgi to the ER. The present study delivers evidence for cytosolic co-localization of both BAP31 and CFTR and for the control of expression of recombinant CFTR in Chinese hamster ovary (CHO) cells and Xenopus oocytes by BAP31. Antisense inhibition of BAP31 in various cell types increased expression of both wild-type CFTR and [DeltaPhe(508)]CFTR and enabled cAMP-activated Cl(-) currents in [DeltaPhe(508)]CFTR-expressing CHO cells. Coexpression of CFTR together with BAP31 attenuated cAMP-activated Cl(-) currents in Xenopus oocytes. These data therefore suggest association of BAP31 with CFTR that may control maturation or trafficking of CFTR and thus expression in the plasma membrane.

  11. A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine

    DEFF Research Database (Denmark)

    Nielsen, Alex Toftgaard; Dolganov, N. A.; Rasmussen, Thomas

    2010-01-01

    A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene...... expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co...... into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients....

  12. Prediction of Children's Empathy-Related Responding from Their Effortful Control and Parents' Expressivity

    Science.gov (United States)

    Valiente, Carlos; Eisenberg, Nancy; Fabes, Richard A.; Shepard, Stephanie A.; Cumberland, Amanda; Losoya, Sandra H.

    2004-01-01

    In this study, the linear and interactive relations of children's effortful control and parents' emotional expressivity to children's empathy-related responses were examined. Participants were 214 children, 4.5 to 8 years old. Children's effortful control was negatively related to their personal distress and was positively related to their…

  13. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes

    DEFF Research Database (Denmark)

    Ventre, I.; Goodman, A.L.; Vallet-Gely, I.

    2006-01-01

    S, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharicles. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge...... by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection...

  14. Impaired T cell responsiveness to interleukin-6 in hematological patients with invasive aspergillosis.

    Directory of Open Access Journals (Sweden)

    Jose F Camargo

    Full Text Available Invasive mold infections (IMI are among the most devastating complications following chemotherapy and hematopoietic stem cell transplantation (HSCT, with high mortality rates. Yet, the molecular basis for human susceptibility to invasive aspergillosis (IA and mucormycosis remain poorly understood. Herein, we aimed to characterize the immune profile of individuals with hematological malignancies (n = 18 who developed IMI during the course of chemotherapy or HSCT, and compared it to that of hematological patients who had no evidence of invasive fungal infection (n = 16. First, we measured the expression of the pattern recognition receptors pentraxin 3, dectin-1, and Toll-like receptors (TLR 2 and 4 in peripheral blood of chemotherapy and HSCT recipients with IMI. Compared to hematological controls, individuals with IA and mucormycosis had defective expression of dectin-1; in addition, patients with mucormycosis had decreased TLR2 and increased TLR4 expression. Since fungal recognition via dectin-1 favors T helper 17 responses and the latter are highly dependent on activation of the signal transducer and activator of transcription (STAT 3, we next used phospho-flow cytometry to measure the phosphorylation of the transcription factors STAT1 and STAT3 in response to interferon-gamma (IFN-γ and interleukin (IL-6, respectively. While IFN-γ/STAT1 signaling was similar between groups, naïve T cells from patients with IA, but not those with mucormycosis, exhibited reduced responsiveness to IL-6 as measured by STAT3 phosphorylation. Furthermore, IL-6 increased Aspergillus-induced IL-17 production in culture supernatants from healthy and hematological controls but not in patients with IA. Altogether, these observations suggest an important role for dectin-1 and the IL-6/STAT3 pathway in protective immunity against Aspergillus.

  15. Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor.

    Science.gov (United States)

    Ryan, J J; McReynolds, L J; Keegan, A; Wang, L H; Garfein, E; Rothman, P; Nelms, K; Paul, W E

    1996-02-01

    IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

  16. [Proteolytic control of expression of Vibrio fischeri lux-operon genes in Escherichia coli cells].

    Science.gov (United States)

    Mel'kina, O E; Manukhov, I V; Zavil'gel'skiĭ, G B

    2010-08-01

    The key elements of the regulatory system activating expression of the lux-operon genes in the sea bacteria Vibrio fischeri are the LuxR protein (an activator oftranscription) and N-(3-oxohexanoyl) L-homoserine lactone (an autoinducer, AI). It is shown that the ATP-dependent proteases ClpXP and Lon take part in the negative control of expression of the lux-operon genes and that AI protects the LuxR protein from proteolysis.

  17. Mosquito Passage Dramatically Changes var Gene Expression in Controlled Human Plasmodium falciparum Infections.

    Science.gov (United States)

    Bachmann, Anna; Petter, Michaela; Krumkamp, Ralf; Esen, Meral; Held, Jana; Scholz, Judith A M; Li, Tao; Sim, B Kim Lee; Hoffman, Stephen L; Kremsner, Peter G; Mordmüller, Benjamin; Duffy, Michael F; Tannich, Egbert

    2016-04-01

    Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase.

  18. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.

    Directory of Open Access Journals (Sweden)

    Alex T Nielsen

    Full Text Available A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP and cholera toxin (CT were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a

  19. MultiRankSeq: multiperspective approach for RNAseq differential expression analysis and quality control.

    Science.gov (United States)

    Guo, Yan; Zhao, Shilin; Ye, Fei; Sheng, Quanhu; Shyr, Yu

    2014-01-01

    After a decade of microarray technology dominating the field of high-throughput gene expression profiling, the introduction of RNAseq has revolutionized gene expression research. While RNAseq provides more abundant information than microarray, its analysis has proved considerably more complicated. To date, no consensus has been reached on the best approach for RNAseq-based differential expression analysis. Not surprisingly, different studies have drawn different conclusions as to the best approach to identify differentially expressed genes based upon their own criteria and scenarios considered. Furthermore, the lack of effective quality control may lead to misleading results interpretation and erroneous conclusions. To solve these aforementioned problems, we propose a simple yet safe and practical rank-sum approach for RNAseq-based differential gene expression analysis named MultiRankSeq. MultiRankSeq first performs quality control assessment. For data meeting the quality control criteria, MultiRankSeq compares the study groups using several of the most commonly applied analytical methods and combines their results to generate a new rank-sum interpretation. MultiRankSeq provides a unique analysis approach to RNAseq differential expression analysis. MultiRankSeq is written in R, and it is easily applicable. Detailed graphical and tabular analysis reports can be generated with a single command line.

  20. tCRISPRi: tunable and reversible, one-step control of gene expression

    Science.gov (United States)

    Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

    2016-12-01

    The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

  1. Gene expression analysis in prostate cancer: the importance of the endogenous control.

    LENUS (Irish Health Repository)

    Vajda, Alice

    2013-03-01

    Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer.

  2. Neural Correlates of Conflict Control on Facial Expressions with a Flanker Paradigm

    DEFF Research Database (Denmark)

    Liu, T.; Xiao, T; Shi, Jiannong

    2013-01-01

    results manifested that participants exhibited faster response speed in identifying neutral target face when it was flanked by neutral distractors than by happy distractors. Electrophysiological results showed that happy target expression induced larger N2 amplitude when flanked by sad distractors than...... by happy distractors and scramble blocks during the conflict monitoring processing. During the attentional control processing, happy target expression induced faster P3 response when it was flanked by happy distractors than by sad distractors, and sad target expression evoked larger P3 amplitude when...... it was flanked by happy distractors comparing with sad distractors. Taken together, the current findings of temporal dynamic of brain activity during cognitive control on affective conflicts shed light on the essential relationship between cognitive control and affective information processing....

  3. Selective Control of Fear Expression by Optogenetic Manipulation of Infralimbic Cortex after Extinction.

    Science.gov (United States)

    Kim, Hyung-Su; Cho, Hye-Yeon; Augustine, George J; Han, Jin-Hee

    2016-04-01

    Evidence from rodent and human studies has identified the ventromedial prefrontal cortex, specifically the infralimbic cortex (IL), as a critical brain structure in the extinction of conditioned fear. However, how IL activity controls fear expression at the time of extinction memory retrieval is unclear and controversial. To address this issue, we used optogenetics to precisely manipulate the activity of genetically targeted cells and to examine the real-time contribution of IL activity to expression of auditory-conditioned fear extinction in mice. We found that inactivation of IL, but not prelimbic cortex, impaired extinction retrieval. Conversely, photostimulation of IL excitatory neurons robustly enhanced the inhibition of fear expression after extinction, but not before extinction. Moreover, this effect was specific to the conditioned stimulus (CS): IL activity had no effect on expression of fear in response to the conditioned context after auditory fear extinction. Thus, in contrast to the expectation from a generally held view, artificial activation of IL produced no significant effect on expression of non-extinguished conditioned fear. Therefore, our data provide compelling evidence that IL activity is critical for expression of fear extinction and establish a causal role for IL activity in controlling fear expression in a CS-specific manner after extinction.

  4. Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition.

    Science.gov (United States)

    Diepenbruck, Maren; Waldmeier, Lorenz; Ivanek, Robert; Berninger, Philipp; Arnold, Phil; van Nimwegen, Erik; Christofori, Gerhard

    2014-04-01

    The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.

  5. Attentional control and interpretation of facial expression after oxytocin administration to typically developed male adults.

    Science.gov (United States)

    Hirosawa, Tetsu; Kikuchi, Mitsuru; Okumura, Eiichi; Yoshimura, Yuko; Hiraishi, Hirotoshi; Munesue, Toshio; Takesaki, Natsumi; Furutani, Naoki; Ono, Yasuki; Higashida, Haruhiro; Minabe, Yoshio

    2015-01-01

    Deficits in attentional-inhibitory control have been reported to correlate to anger, hostility, and aggressive behavior; therefore, inhibitory control appears to play an important role in prosocial behavior. Moreover, recent studies have demonstrated that oxytocin (OT) exerts a prosocial effect (e.g., decreasing negative behaviors, such as aggression) on humans. However, it is unknown whether the positively valenced effect of OT on sociality is associated with enhanced attentional-inhibitory control. In the present study, we hypothesized that OT enhances attentional-inhibitory control and that the positively valenced effect of OT on social cognition is associated with enhanced attentional-inhibitory control. In a single-blind, placebo-controlled crossover trial, we tested this hypothesis using 20 healthy male volunteers. We considered a decrease in the hostility detection ratio, which reflects the positively valenced interpretation of other individuals' facial expressions, to be an index of the positively valenced effects of OT (we reused the results of our previously published study). As a measure of attentional-inhibitory control, we employed a modified version of the flanker task (i.e., a shorter conflict duration indicated higher inhibitory control). These results failed to demonstrate any significant behavioral effects of OT (i.e., neither a positively valenced effect on facial cognition nor an effect on attentional-inhibitory control). However, the enhancement of attentional-inhibitory control after OT administration significantly correlated to the positively valenced effects on the interpretation of uncertain facial cognition (i.e., neutral and ambiguous facial expressions).

  6. An express method for optimally tuning an analog controller with respect to integral quality criteria

    Science.gov (United States)

    Golinko, I. M.; Kovrigo, Yu. M.; Kubrak, A. I.

    2014-03-01

    An express method for optimally tuning analog PI and PID controllers is considered. An integral quality criterion with minimizing the control output is proposed for optimizing control systems. The suggested criterion differs from existing ones in that the control output applied to the technological process is taken into account in a correct manner, due to which it becomes possible to maximally reduce the expenditure of material and/or energy resources in performing control of industrial equipment sets. With control organized in such manner, smaller wear and longer service life of control devices are achieved. A unimodal nature of the proposed criterion for optimally tuning a controller is numerically demonstrated using the methods of optimization theory. A functional interrelation between the optimal controller parameters and dynamic properties of a controlled plant is numerically determined for a single-loop control system. The results obtained from simulation of transients in a control system carried out using the proposed and existing functional dependences are compared with each other. The proposed calculation formulas differ from the existing ones by a simple structure and highly accurate search for the optimal controller tuning parameters. The obtained calculation formulas are recommended for being used by specialists in automation for design and optimization of control systems.

  7. Control of the expression of anchored genes using micron scale heater

    Science.gov (United States)

    Shivashankar, G. V.; Liu, S.; Libchaber, A.

    2000-06-01

    We control in vitro gene expression, using DNA (gene) sequences immobilized on miniaturized heaters. For this, electronically addressable indium-tin-oxide heating pads, 100 μm2 in size, are fabricated on a glass substrate to locally define temperatures and thus control transcription and translation of DNA gene sequences attached to the pad. Wheat germ cell extract is the reaction medium. We use luciferase gene and monitor its temperature-controlled expression by luminescence. Various constructs are tested yielding either luciferase free or bound to the coding DNA. Control of protein production at precise sites and rates, using temperature, opens the possibility to engineer various protein networks with different time scales. Working with constructs where the protein stays bound, gene product micro-arrays can be realized.

  8. Control of Neuropeptide Expression by Parallel Activity-dependent Pathways in Caenorhabditis elegans

    Science.gov (United States)

    Rojo Romanos, Teresa; Petersen, Jakob Gramstrup; Pocock, Roger

    2017-01-01

    Monitoring of neuronal activity within circuits facilitates integrated responses and rapid changes in behavior. We have identified a system in Caenorhabditis elegans where neuropeptide expression is dependent on the ability of the BAG neurons to sense carbon dioxide. In C. elegans, CO2 sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO2 causes accumulation of cyclic GMP and opening of the cGMP-gated TAX-2/TAX-4 cation channels; provoking an integrated downstream cascade that enables C. elegans to avoid high CO2. Here we show that cGMP regulation by GCY-9 and the PDE-1 phosphodiesterase controls BAG expression of a FMRFamide-related neuropeptide FLP-19 reporter (flp-19::GFP). This regulation is specific for CO2-sensing function of the BAG neurons, as loss of oxygen sensing function does not affect flp-19::GFP expression. We also found that expression of flp-19::GFP is controlled in parallel to GCY-9 by the activity-dependent transcription factor CREB (CRH-1) and the cAMP-dependent protein kinase (KIN-2) signaling pathway. We therefore show that two parallel pathways regulate neuropeptide gene expression in the BAG sensory neurons: the ability to sense changes in carbon dioxide and CREB transcription factor. Such regulation may be required in particular environmental conditions to enable sophisticated behavioral decisions to be performed. PMID:28139692

  9. Gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mRNA degradation.

    Directory of Open Access Journals (Sweden)

    Emma Abernathy

    2014-01-01

    Full Text Available Lytic gammaherpesvirus infection restricts host gene expression by promoting widespread degradation of cytoplasmic mRNA through the activity of the viral endonuclease SOX. Though generally assumed to be selective for cellular transcripts, the extent to which SOX impacts viral mRNA stability has remained unknown. We addressed this issue using the model murine gammaherpesvirus MHV68 and, unexpectedly, found that all stages of viral gene expression are controlled through mRNA degradation. Using both comprehensive RNA expression profiling and half-life studies we reveal that the levels of the majority of viral mRNAs but not noncoding RNAs are tempered by MHV68 SOX (muSOX activity. The targeting of viral mRNA by muSOX is functionally significant, as it impacts intracellular viral protein abundance and progeny virion composition. In the absence of muSOX-imposed gene expression control the viral particles display increased cell surface binding and entry as well as enhanced immediate early gene expression. These phenotypes culminate in a viral replication defect in multiple cell types as well as in vivo, highlighting the importance of maintaining the appropriate balance of viral RNA during gammaherpesviral infection. This is the first example of a virus that fails to broadly discriminate between cellular and viral transcripts during host shutoff and instead uses the targeting of viral messages to fine-tune overall gene expression.

  10. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses

    Directory of Open Access Journals (Sweden)

    Ieisha Pentland

    2015-07-01

    Full Text Available All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV, Epstein-Barr virus (EBV and human papillomavirus (HPV utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  11. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    Science.gov (United States)

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  12. Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia.

    Science.gov (United States)

    Rana, Sobia; Munawar, Mustafa; Shahid, Adeela; Malik, Meera; Ullah, Hafeez; Fatima, Warda; Mohsin, Shahida; Mahmood, Saqib

    2014-01-01

    Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.

  13. Expressed breast milk on a neonatal unit: a hazard analysis and critical control points approach.

    Science.gov (United States)

    Cossey, Veerle; Jeurissen, Axel; Thelissen, Marie-José; Vanhole, Chris; Schuermans, Annette

    2011-12-01

    With the increasing use of human milk and growing evidence of the benefits of mother's milk for preterm and ill newborns, guidelines to ensure its quality and safety are an important part of daily practice in neonatal intensive care units. Operating procedures based on hazard analysis and critical control points can standardize the handling of mother's expressed milk, thereby improving nutrition and minimizing the risk of breast milk-induced infection in susceptible newborns. Because breast milk is not sterile, microorganisms can multiply when the milk is not handled properly. Additional exogenous contamination should be prevented. Strict hygiene and careful temperature and time control are important during the expression, collection, transport, storage, and feeding of maternal milk. In contrast to formula milk, no legal standards exist for the use of expressed maternal milk. The need for additional measures, such as bacteriological screening or heat treatment, remains unresolved.

  14. Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch.

    Science.gov (United States)

    Ramundo, Silvia; Rochaix, Jean-David

    2015-01-01

    Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressible and reversible expression systems are highly desirable for functional and biochemical characterization of these genes. We have developed a genetic tool that allows one to regulate the expression of any coding sequence in the chloroplast genome of the unicellular alga Chlamydomonas reinhardtii. Our system is based on vitamin-regulated expression of the nucleus-encoded chloroplast Nac2 protein, which is specifically required for the expression of any plastid gene fused to the psbD 5'UTR. With this approach, expression of the Nac2 gene in the nucleus and, in turn, that of the chosen chloroplast gene artificially driven by the psbD 5'UTR, is controlled by the MetE promoter and Thi4 riboswitch, which can be inactivated in a reversible way by supplying vitamin B12 and thiamine to the growth medium, respectively. This system opens interesting possibilities for studying the assembly and turnover of chloroplast multiprotein complexes such as the photosystems, the ribosome, and the RNA polymerase. It also provides a way to overcome the toxicity often associated with the expression of proteins of biotechnological interest in the chloroplast.

  15. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes

    DEFF Research Database (Denmark)

    Ventre, I.; Goodman, A.L.; Vallet-Gely, I.

    2006-01-01

    by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection...

  16. Emotional Expression and Control in School-Age Children in India and the United States

    Science.gov (United States)

    Wilson, Stephanie L.; Raval, Vaishali V.; Salvina, Jennifer; Raval, Pratiksha H.; Panchal, Ila N.

    2012-01-01

    The present study compared 6- to 9-year-old children's reports of their decisions to express anger, sadness, and physical pain; methods of controlling and communicating felt emotion; and reasons for doing so in response to hypothetical situations across three groups: old-city India (n = 60), suburban India (n = 60), and suburban United States (n =…

  17. Dissecting Daily and Circadian Expression Rhythms of Clock-Controlled Genes in Human Blood

    NARCIS (Netherlands)

    K. Lech (Karolina); K. Ackermann (Katrin); V.L. Revell (Victoria); O.S.C.A.R. Lao; D.J. Skene (Debra); M.H. Kayser (Manfred)

    2016-01-01

    textabstractThe identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human

  18. Dissecting the logical types of network control in gene expression profiles

    Directory of Open Access Journals (Sweden)

    Geertz Marcel

    2008-02-01

    Full Text Available Abstract Background In the bacterium Escherichia coli the transcriptional regulation of gene expression involves both dedicated regulators binding specific DNA sites with high affinity and also global regulators – abundant DNA architectural proteins of the bacterial nucleoid binding multiple sites with a wide range of affinities and thus modulating the superhelical density of DNA. The first form of transcriptional regulation is predominantly pairwise and specific, representing digitial control, while the second form is (in strength and distribution continuous, representing analog control. Results Here we look at the properties of effective networks derived from significant gene expression changes under variation of the two forms of control and find that upon limitations of one type of control (caused e.g. by mutation of a global DNA architectural factor the other type can compensate for compromised regulation. Mutations of global regulators significantly enhance the digital control, whereas in the presence of global DNA architectural proteins regulation is mostly of the analog type, coupling spatially neighboring genomic loci. Taken together our data suggest that two logically distinct – digital and analog – types of control are balancing each other. Conclusion By revealing two distinct logical types of control, our approach provides basic insights into both the organizational principles of transcriptional regulation and the mechanisms buffering genetic flexibility. We anticipate that the general concept of distinguishing logical types of control will apply to many complex biological networks.

  19. Development of gene microarray in screening differently expressed genes in keloid and normal-control skin

    Institute of Scientific and Technical Information of China (English)

    陈伟; 付小兵; 葛世丽; 孙晓庆; 周岗; 赵志力; 盛志勇

    2004-01-01

    Background Keloid is an intricate lesion that is probably regulated by many genes. In this study, the authors used the technique of complementary DNA (cDNA) microarray to analyse abnormal gene expression in keloids and normal control skins. Methods The polymerase chain reaction (PCR) products of 8400 genes were spotted in an array on chemical-material-coated-glass plates. The DNAs were fixed on the glass plates. The total RNAs were isolated from freshly excised human keloid and normal control skins, and the mRNAs were then purified. The mRNA from both keloid and normal control skins were reversely transcribed to cDNAs, with the incorporation of fluorescent dUTP, for preparing the hybridisation probes. The mixed probes were then hybridised to the cDNA microarray. After thorough washing, the cDNA microarray was scanned for differing fluorescent signals from two types of tissues. Gene expression of tissue growth factor-β1 (TGF-β1) and of c-myc was detected with both RT-PCR and Northern blot hybridisation to confirm the effectiveness of cDNA microarray. Results Among the 8400 human genes, 402 were detected with different expression levels between keloid and normal control skins. Two hundred and fifty genes, including TGF-β1 and c-myc, were up-regulated and 152 genes were down-regulated. Higher expressions of TGF-β1 and c-myc in keloid were also revealed using RT-PCR and Northern blot methods. Conclusion cDNA microarray analysis provides a powerful tool for investigating differential gene expression in keloid and normal control skins. Keloid is a complicated lesion with many genes involved.

  20. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Science.gov (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  1. Effect of TPA and HTLV-1 Tax on BRCA1 and ERE controlled genes expression.

    Science.gov (United States)

    Jabareen, Azhar; Abu-Jaafar, Aya; Abou-Kandil, Ammar; Huleihel, Mahmoud

    2017-07-18

    Interference with the expression and/or functions of the multifunctional tumor suppressor BRCA1 leads to a high risk of breast and ovarian cancers. BRCA1 expression is usually activated by the estrogen (E2) liganded ERα receptor. Activated ERα is considered as a potent transcription factor which activates various genes expression by 2 pathways. A classical pathway, ERα binds directly to E2-responsive elements (EREs) in the promoters of the responsive genes and a non-classical pathway where ERα indirectly binds with the appropriate gene promoter. In our previous study, HTLV-1Tax was found to strongly inhibit ERα induced BRCA1 expression while stimulating ERα induced ERE dependent genes. TPA is a strong PKC activator which found to induce the expression of HTLV-1. Here we examined the effect of TPA on the expression of BRCA1 and genes controlled by ERE region in MCF-7 cells and on Tax activity on these genes. Our results showed strong stimulatory effect of TPA on both BRCA1 and ERE expression without treatment with E2. Tax did not show any significant effect on these TPA activities. It seems that TPA activation of BRCA1 and ERE expression is dependent on PKC activity but not through the NFκB pathway. However, 53BP1 may be involved in this TPA activity because its overexpression significantly reduced the TPA stimulatory effect on BRCA1 and ERE expression. Additionally, our Chip assay results probably exclude possible involvement of ERα pathway in this TPA activity because TPA did not interfere with the binding of ERα to both BRCA1 promoter and ERE region.

  2. Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth

    Science.gov (United States)

    Milias-Argeitis, Andreas; Rullan, Marc; Aoki, Stephanie K.; Buchmann, Peter; Khammash, Mustafa

    2016-01-01

    Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology. PMID:27562138

  3. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  4. Transcriptional Mechanisms Controlling miR-375 Gene Expression in the Pancreas

    Directory of Open Access Journals (Sweden)

    Tali Avnit-Sagi

    2012-01-01

    Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that play an important role in mediating a broad and expanding range of biological activities. miR-375 is expressed selectively in the pancreas. We have previously shown that selective expression of miR-375 in pancreatic beta cells is controlled by transcriptional mechanisms operating through a TATA box-containing promoter. Expression of miR-375 has been reported in non-beta cells within the endocrine pancreas, and indeed inactivation of miR-375 leads to perturbation in cell mass and number of both alpha and beta cells. Consistent with its expression throughout the endocrine pancreas, we now show that the promoter of the miR-375 gene shows selective activity in pancreatic endocrine alpha cells, comparable to that observed in beta cells. We previously identified a novel negative regulatory element located downstream of the miR-375 gene transcription start site. By generating luciferase reporter genes, we now show that the sequence is functional also when positioned upstream of a heterologous promoter, thus proving that the repressor effect is mediated at least in part at the level of transcription. Further characterization of the transcriptional control mechanism regulating expression of miR-375 and other pancreatic miRNAs will contribute to a better understanding of pancreas development and function.

  5. Fine tuning of IRF-4 expression by SWAP-70 controls the initiation of plasma cell development.

    Science.gov (United States)

    Chopin, Michaël; Chacón-Martínez, Carlos Andrés; Jessberger, Rolf

    2011-10-01

    The generation of plasma cells (PCs) is key for proper humoral immune responses. The transcription factors IRF-4 and BLIMP-1 (B-lymphocyte induce maturation protein-1) control PC commitment, but the underlying regulatory mechanisms are incompletely understood. Here we have identified SWAP-70 as being critically involved in Toll-like receptor (TLR)-triggered PC differentiation. Upon activation through various TLRs, Swap-70(-/-) B cells were activated and proliferated normally. However, expression of BLIMP-1 was markedly reduced and PC differentiation was impaired. Four hours of LPS stimulation were sufficient to drive PC differentiation, and SWAP-70 was required during this initial period. Swap-70(-/-) B cells pre-activated in vitro failed to efficiently differentiate into PCs upon adoptive transfer into recipient mice. Re-introduction of SWAP-70 into Swap-70(-/-) B cells rescued their development into PCs, and SWAP-70 over-expression in wild-type (WT) B cells increased PC generation. In the absence of SWAP-70, IRF-4 protein levels were reduced and the IRF-4(high) B220(+) CD138(-) compartment, including PC precursors, was strongly diminished. Ectopic expression of SWAP-70 increases IRF-4 protein levels and PC differentiation in WT and Swap-70(-/-) B cells, and IRF-4 over-expression in Swap-70(-/-) B cells elevates PC differentiation to WT levels. Thus, in a dose-dependent manner, SWAP-70 controls IRF-4 protein expression and thereby regulates the initiation of PC differentiation.

  6. A synthetic library of RNA control modules for predictable tuning of gene expression in yeast.

    Science.gov (United States)

    Babiskin, Andrew H; Smolke, Christina D

    2011-03-01

    Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae. A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems.

  7. ERECTA signaling controls Arabidopsis inflorescence architecture through chromatin-mediated activation of PRE1 expression.

    Science.gov (United States)

    Cai, Hanyang; Zhao, Lihua; Wang, Lulu; Zhang, Man; Su, Zhenxia; Cheng, Yan; Zhao, Heming; Qin, Yuan

    2017-03-13

    Flowering plants display a remarkable diversity in inflorescence architecture, and pedicel length is one of the key contributors to this diversity. In Arabidopsis thaliana, the receptor-like kinase ERECTA (ER) mediated signaling pathway plays important roles in regulating inflorescence architecture by promoting cell proliferation. However, the regulating mechanism remains elusive in the pedicel. Genetic interactions between ERECTA signaling and the chromatin remodeling complex SWR1 in the control of inflorescence architecture were studied. Comparative transcriptome analysis was applied to identify downstream components. Chromatin immunoprecipitation and nucleosome occupancy was further investigated. The results indicated that the chromatin remodeler SWR1 coordinates with ERECTA signaling in regulating inflorescence architecture by activating the expression of PRE1 family genes and promoting pedicel elongation. It was found that SWR1 is required for the incorporation of the H2A.Z histone variant into nucleosomes of the whole PRE1 gene family and the ERECTA controlled expression of PRE1 gene family through regulating nucleosome dynamics. We propose that utilization of a chromatin remodeling complex to regulate gene expression is a common theme in developmental control across kingdoms. These findings shed light on the mechanisms through which chromatin remodelers orchestrate complex transcriptional regulation of gene expression in coordination with a developmental cue.

  8. A Biogenesis Step Upstream of Microprocessor Controls miR-17∼92 Expression.

    Science.gov (United States)

    Du, Peng; Wang, Longfei; Sliz, Piotr; Gregory, Richard I

    2015-08-13

    The precise control of miR-17∼92 microRNA (miRNA) is essential for normal development, and overexpression of certain miRNAs from this cluster is oncogenic. Here, we find that the relative expression of the six miRNAs processed from the primary (pri-miR-17∼92) transcript is dynamically regulated during embryonic stem cell (ESC) differentiation. Pri-miR-17∼92 is processed to a biogenesis intermediate, termed "progenitor-miRNA" (pro-miRNA). Pro-miRNA is an efficient substrate for Microprocessor and is required to selectively license production of pre-miR-17, pre-miR-18a, pre-miR-19a, pre-miR-20a, and pre-miR-19b from this cluster. Two complementary cis-regulatory repression domains within pri-miR-17∼92 are required for the blockade of miRNA processing through the formation of an autoinhibitory RNA conformation. The endonuclease CPSF3 (CPSF73) and the spliceosome-associated ISY1 are responsible for pro-miRNA biogenesis and expression of all miRNAs within the cluster except miR-92. Thus, developmentally regulated pro-miRNA processing is a key step controlling miRNA expression and explains the posttranscriptional control of miR-17∼92 expression in development.

  9. Inducible T-cell receptor expression in precursor T-cells for leukemia control

    Science.gov (United States)

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.

    2015-01-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  10. Methotrexate Increases Skeletal Muscle GLUT4 Expression and Improves Metabolic Control in Experimental Diabetes

    Directory of Open Access Journals (Sweden)

    Giuseppina T. Russo

    2012-01-01

    Full Text Available Long-term administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR mimics the effects of endurance exercise by activating AMP kinase and by increasing skeletal muscle expression of GLUT4 glucose transporter. AICAR is an intermediate in the purine de novo synthesis, and its tissue concentrations can be increased, in vivo, by low doses of methotrexate (MTX through the inhibition of the enzyme AICAR transformylase. We report here the first evidence that, in experimental type 2 diabetes, chronic treatment with low doses of MTX increases skeletal muscle GLUT4 expression and improves metabolic control. MTX (0.5 mg/kg body weight or vehicle was administered intraperitoneally, once a week for 4 weeks, to genetically diabetic female C57BL/KsJ-m+/+Leptdb mice (db+/db+ and their normoglycemic littermates (db+/+m. In the db+/db+ mice, MTX treatment was associated with a ∼2-fold increase in skeletal muscle GLUT4 protein concentration and a >4-fold increase in GLUT4 mRNA expression (P<0.01, all, as compared to vehicle-treated mice; no significant differences were noted in controls. MTX treatment was also associated with a significant reduction of glucose and insulin serum concentrations in diabetic mice (P<0.001, and glucose levels only (P<0.05 in controls. These data indicate a different route to increase skeletal muscle GLUT4 expression, through the potential inhibition of the enzyme AICAR transformylase.

  11. A SINGLE TETRACYCLINE-REGULATED VECTOR DEVISED FOR CONTROLLED INSULIN GENE EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    Xue-yang Zhang; Ben-li Su; Hong Li; Ran Bai; Zhao-hui Xu; Chang-chen Li

    2004-01-01

    Objective To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line.Methods An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recom bined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 μg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day.Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits.Results Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation,and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day.Conclusion Human insulin expression can be successfully regulated by doxycycline and the background was very low.This single ret-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.

  12. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    Science.gov (United States)

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

  13. MiRNA-mediated control of HLA-G expression and function.

    Directory of Open Access Journals (Sweden)

    Irit Manaster

    Full Text Available HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3'UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3'UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G.

  14. A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters

    Science.gov (United States)

    Javaid, Shaista; Amin, Imran; Jander, Georg; Mukhtar, Zahid; Saeed, Nasir A.; Mansoor, Shahid

    2016-01-01

    The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests. PMID:27708374

  15. Islet amyloid polypeptide and insulin expression are controlled differently in primary and transformed islet cells

    DEFF Research Database (Denmark)

    Madsen, O D; Michelsen, Bo Thomas; Westermark, P;

    1991-01-01

    in unstable heterogeneous clones such as NHI-6F. This clone is composed of primarily glucagon-producing cells in vitro, but insulin gene expression becomes dominant after passage in vivo. Interestingly, IAPP was hyperexpressed with glucagon under in vitro conditions in this clone. We conclude that the tissue...... specificity of expressions of IAPP and insulin are controlled differently, and that coexpression of IAPP with hormones different from insulin may be a marker for pluripotent transformed rat islet cell clones, which are able to activate insulin gene transcription during passage in vivo....

  16. Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene.

    OpenAIRE

    Guarneros, G; Montañez, C; Hernandez, T; Court, D

    1982-01-01

    The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from t...

  17. Conserved Cis-Regulatory Modules Control Robustness in Msx1 Expression at Single-Cell Resolution

    Science.gov (United States)

    Vance, Keith W.; Woodcock, Dan J.; Reid, John E.; Bretschneider, Till; Ott, Sascha; Koentges, Georgy

    2015-01-01

    The process of transcription is highly stochastic leading to cell-to-cell variations and noise in gene expression levels. However, key essential genes have to be precisely expressed at the correct amount and time to ensure proper cellular development and function. Studies in yeast and bacterial systems have shown that gene expression noise decreases as mean expression levels increase, a relationship that is controlled by promoter DNA sequence. However, the function of distal cis-regulatory modules (CRMs), an evolutionary novelty of metazoans, in controlling transcriptional robustness and variability is poorly understood. In this study, we used live cell imaging of transfected reporters combined with a mathematical modelling and statistical inference scheme to quantify the function of conserved Msx1 CRMs and promoters in modulating single-cell real-time transcription rates in C2C12 mouse myoblasts. The results show that the mean expression–noise relationship is solely promoter controlled for this key pluripotency regulator. In addition, we demonstrate that CRMs modulate single-cell basal promoter rate distributions in a graded manner across a population of cells. This extends the rheostatic model of CRM action to provide a more detailed understanding of CRM function at single-cell resolution. We also identify a novel CRM transcriptional filter function that acts to reduce intracellular variability in transcription rates and show that this can be phylogenetically separable from rate modulating CRM activities. These results are important for understanding how the expression of key vertebrate developmental transcription factors is precisely controlled both within and between individual cells. PMID:26342140

  18. Detection of RUNX2 gene expression in cumulus cells in women undergoing controlled ovarian stimulation

    Directory of Open Access Journals (Sweden)

    Papamentzelopoulou Myrto

    2012-11-01

    Full Text Available Abstract Background RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. Methods A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. Results Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0. Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52. From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013, higher number of retrieved oocytes (p = 0.016, higher basal LH serum levels (p = 0.016 and higher peak estradiol levels (p = 0.013, while the number of fertilized oocytes differed marginally between the two groups (p = 0.089. Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021 and E2 levels (OR = 0.25, p = 0.026. Conclusions Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.

  19. Assessment of CcpA-mediated catabolite control of gene expression in Bacillus cereus ATCC 14579

    Directory of Open Access Journals (Sweden)

    Buist Girbe

    2008-04-01

    Full Text Available Abstract Background The catabolite control protein CcpA is a transcriptional regulator conserved in many Gram-positives, controlling the efficiency of glucose metabolism. Here we studied the role of Bacillus cereus ATCC 14579 CcpA in regulation of metabolic pathways and expression of enterotoxin genes by comparative transcriptome analysis of the wild-type and a ccpA-deletion strain. Results Comparative analysis revealed the growth performance and glucose consumption rates to be lower in the B. cereus ATCC 14579 ccpA deletion strain than in the wild-type. In exponentially grown cells, the expression of glycolytic genes, including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, was down-regulated and expression of gluconeogenic genes and genes encoding the citric acid cycle was up-regulated in the B. cereus ccpA deletion strain. Furthermore, putative CRE-sites, that act as binding sites for CcpA, were identified to be present for these genes. These results indicate CcpA to be involved in the regulation of glucose metabolism, thereby optimizing the efficiency of glucose catabolism. Other genes of which the expression was affected by ccpA deletion and for which putative CRE-sites could be identified, included genes with an annotated function in the catabolism of ribose, histidine and possibly fucose/arabinose and aspartate. Notably, expression of the operons encoding non-hemolytic enterotoxin (Nhe and hemolytic enterotoxin (Hbl was affected by ccpA deletion, and putative CRE-sites were identified, which suggests catabolite repression of the enterotoxin operons to be CcpA-dependent. Conclusion The catabolite control protein CcpA in B. cereus ATCC 14579 is involved in optimizing the catabolism of glucose with concomitant repression of gluconeogenesis and alternative metabolic pathways. Furthermore, the results point to metabolic control

  20. Attentional Control and Interpretation of Facial Expression after Oxytocin Administration to Typically Developed Male Adults

    Science.gov (United States)

    Hirosawa, Tetsu; Kikuchi, Mitsuru; Okumura, Eiichi; Yoshimura, Yuko; Hiraishi, Hirotoshi; Munesue, Toshio; Takesaki, Natsumi; Furutani, Naoki; Ono, Yasuki; Higashida, Haruhiro; Minabe, Yoshio

    2015-01-01

    Deficits in attentional-inhibitory control have been reported to correlate to anger, hostility, and aggressive behavior; therefore, inhibitory control appears to play an important role in prosocial behavior. Moreover, recent studies have demonstrated that oxytocin (OT) exerts a prosocial effect (e.g., decreasing negative behaviors, such as aggression) on humans. However, it is unknown whether the positively valenced effect of OT on sociality is associated with enhanced attentional-inhibitory control. In the present study, we hypothesized that OT enhances attentional-inhibitory control and that the positively valenced effect of OT on social cognition is associated with enhanced attentional-inhibitory control. In a single-blind, placebo-controlled crossover trial, we tested this hypothesis using 20 healthy male volunteers. We considered a decrease in the hostility detection ratio, which reflects the positively valenced interpretation of other individuals’ facial expressions, to be an index of the positively valenced effects of OT (we reused the results of our previously published study). As a measure of attentional-inhibitory control, we employed a modified version of the flanker task (i.e., a shorter conflict duration indicated higher inhibitory control). These results failed to demonstrate any significant behavioral effects of OT (i.e., neither a positively valenced effect on facial cognition nor an effect on attentional-inhibitory control). However, the enhancement of attentional-inhibitory control after OT administration significantly correlated to the positively valenced effects on the interpretation of uncertain facial cognition (i.e., neutral and ambiguous facial expressions). PMID:25659131

  1. Attentional control and interpretation of facial expression after oxytocin administration to typically developed male adults.

    Directory of Open Access Journals (Sweden)

    Tetsu Hirosawa

    Full Text Available Deficits in attentional-inhibitory control have been reported to correlate to anger, hostility, and aggressive behavior; therefore, inhibitory control appears to play an important role in prosocial behavior. Moreover, recent studies have demonstrated that oxytocin (OT exerts a prosocial effect (e.g., decreasing negative behaviors, such as aggression on humans. However, it is unknown whether the positively valenced effect of OT on sociality is associated with enhanced attentional-inhibitory control. In the present study, we hypothesized that OT enhances attentional-inhibitory control and that the positively valenced effect of OT on social cognition is associated with enhanced attentional-inhibitory control. In a single-blind, placebo-controlled crossover trial, we tested this hypothesis using 20 healthy male volunteers. We considered a decrease in the hostility detection ratio, which reflects the positively valenced interpretation of other individuals' facial expressions, to be an index of the positively valenced effects of OT (we reused the results of our previously published study. As a measure of attentional-inhibitory control, we employed a modified version of the flanker task (i.e., a shorter conflict duration indicated higher inhibitory control. These results failed to demonstrate any significant behavioral effects of OT (i.e., neither a positively valenced effect on facial cognition nor an effect on attentional-inhibitory control. However, the enhancement of attentional-inhibitory control after OT administration significantly correlated to the positively valenced effects on the interpretation of uncertain facial cognition (i.e., neutral and ambiguous facial expressions.

  2. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

    Science.gov (United States)

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria

    2011-03-01

    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  3. Differential modulation of gene expression in the NMDA postsynaptic density of schizophrenic and control smokers.

    Science.gov (United States)

    Mexal, S; Frank, M; Berger, R; Adams, C E; Ross, R G; Freedman, R; Leonard, S

    2005-10-03

    Nicotine is known to induce the release of multiple neurotransmitters, including glutamate and dopamine, through activation of nicotinic receptors. Gene expression in the N-methyl-d-aspartate postsynaptic density (NMDA-PSD), as well as other functional groups, was compared in postmortem hippocampus of schizophrenic and nonmentally ill smokers and nonsmokers utilizing a microarray and quantitative RT-PCR approach. The expression of 277 genes was significantly changed between all smokers and nonsmokers. Specific gene groups, most notably genes expressed in the NMDA-PSD, were prevalent among these transcripts. Analysis of the interaction between smoking and schizophrenia identified several genes in the NMDA-PSD that were differentially affected by smoking in patients. The present findings suggest that smoking may differentially modulate glutamatergic function in schizophrenic patients and control subjects. The biological mechanisms underlying chronic tobacco use are likely to differ substantially between these two groups.

  4. Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size

    Science.gov (United States)

    Toonen, Ruud F. G.; Wierda, Keimpe; Sons, Michèle S.; de Wit, Heidi; Cornelisse, L. Niels; Brussaard, Arjen; Plomp, Jaap J.; Verhage, Matthijs

    2006-01-01

    Prompt recovery after intense activity is an essential feature of most mammalian synapses. Here we show that synapses with reduced expression of the presynaptic gene munc18-1 suffer from increased depression during intense stimulation at glutamatergic, GABAergic, and neuromuscular synapses. Conversely, munc18-1 overexpression makes these synapses recover faster. Concomitant changes in the readily releasable vesicle pool and its refill kinetics were found. The number of vesicles docked at the active zone and the total number of vesicles per terminal correlated with both munc18-1 expression levels and the size of the releasable vesicle pool. These data show that varying expression of a single gene controls synaptic recovery by modulating the number of docked, release-ready vesicles and thereby replenishment of the secretion capacity. PMID:17110441

  5. Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters.

    Science.gov (United States)

    L'hostis-Guidet, Anne; Recher, Gaëlle; Guillet, Brigitte; Al-Mohammad, Abdulrahim; Coumailleau, Pascal; Tiaho, François; Boujard, Daniel; Madigou, Thierry

    2009-10-01

    Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.

  6. Perpetual expression of PAMPs necessary for optimal immune control and clearance of a persistent pathogen.

    Science.gov (United States)

    Kurup, Samarchith P; Tarleton, Rick L

    2013-01-01

    Pathogen-associated molecular patterns (PAMPs) are known to be fundamental in instigating immune responses, but their role in influencing these responses beyond their initiation is less well understood. Here, using the protozoan parasite Trypanosoma cruzi, which is deficient in strong PAMPs, we demonstrate a requirement for the continuous expression of PAMPs for optimal anti-pathogen immunity. Although co-inoculating with, temporary anchoring of and transgenic expression of exogenous PAMPs all result in enhanced early adaptive immune responses, only the continuous expression of bacterial PAMPs on transgenic T. cruzi sustains these responses, resulting in enhanced pathogen clearance. These findings demonstrate that PAMPs function to potentiate adaptive immune responses well beyond their initiation and may determine the efficiency of control of pathogens capable of long-term persistence.

  7. Anti-microbial peptide gene expression during oral vaccination: analysis of a randomized controlled trial.

    Science.gov (United States)

    Simuyandi, M; Kapulu, M; Kelly, P

    2016-11-01

    We have observed previously that micronutrient supplementation ameliorated suppression of α-defensin expression during diarrhoea. However, how interactions between anti-microbial peptide (AMP) expression and diarrhoeal disease are altered by micronutrient supplementation remain unclear. Using oral vaccination as a model of intestinal infection, we measured changes in AMP expression during multiple micronutrient supplementation. In the first part, volunteers underwent duodenal jejunal biopsy before and at 1, 2, 4 or 7 days after administration of one of three live, attenuated oral vaccines against rotavirus, typhoid and enterotoxigenic Escherichia coli. In the second part, participants were randomized to receive a multiple micronutrient supplement or placebo for 6 weeks before undergoing intestinal biopsy, vaccination against typhoid and rebiopsy after 14 days. Expression of human alpha-defensin (HD)5, HD6, hBD1, hBD2 and LL-37 was measured by quantitative reverse transcription-polymerase chain reaction. Taken together, the bacterial vaccines, but not rotavirus vaccine, reduced HD5 expression (P = 0·02, signed-rank test) and reduced LL-37 expression in seven of the eight individuals whose biopsies had expression prevaccination (P = 0·03). hBD2 was not detected. In the controlled trial, HD5 and HD6 expression after vaccination was lower [median ratio 0·5, interquartile range (IQR) = 0·07-2·2 and 0·58, IQR = 0·13-2·3, respectively] than before vaccination. There was no significant effect detected of micronutrient supplementation on expression of HD5, HD6, hBD1 or LL-37. We conclude that live attenuated bacterial vaccines, but not rotavirus vaccine, can reduce intestinal α-defensins, and typhoid vaccine reduced LL-37 expression. We found no evidence that micronutrient supplementation in the short term had any impact on anti-microbial peptide expression. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd

  8. Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

    Science.gov (United States)

    Geng, Haifeng; Belas, Robert

    2010-09-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

  9. Expression of Tropodithietic Acid Biosynthesis Is Controlled by a Novel Autoinducer▿ †

    Science.gov (United States)

    Geng, Haifeng; Belas, Robert

    2010-01-01

    The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda− mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda− mutants—tdaA and tdaH failed to respond—by placing wild-type (Tda+) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal. PMID:20601479

  10. Runx1 controls terminal morphology and mechanosensitivity of VGLUT3-expressing C-mechanoreceptors.

    Science.gov (United States)

    Lou, Shan; Duan, Bo; Vong, Linh; Lowell, Bradford B; Ma, Qiufu

    2013-01-16

    VGLUT3-expressing unmyelinated low-threshold mechanoreceptors (C-LTMRs) are proposed to mediate pleasant touch and/or pain, but the molecular programs controlling C-LTMR development are unknown. Here, we performed genetic fate mapping, showing that VGLUT3 lineage sensory neurons are divided into two groups, based on transient or persistent VGLUT3 expression. VGLUT3-transient neurons are large- or medium-diameter myelinated mechanoreceptors that form the Merkel cell-neurite complex. VGLUT3-persistent neurons are small-diameter unmyelinated neurons that are further divided into two subtypes: (1) tyrosine hydroxylase (TH)-positive C-LTMRs that form the longitudinal lanceolate endings around hairs, and (2) TH-negative neurons that form epidermal-free nerve endings. We then found that VGLUT3-persistent neurons express the runt domain transcription factor Runx1. Analyses of mice with a conditional knock-out of Runx1 in VGLUT3 lineage neurons demonstrate that Runx1 is pivotal to the development of VGLUT3-persistent neurons, such as the expression of VGLUT3 and TH and the formation of the longitudinal lanceolate endings. Furthermore, Runx1 is required to establish mechanosensitivity in C-LTMRs, by controlling the expression of the mechanically gated ion channel Piezo2. Surprisingly, both acute and chronic mechanical pain was largely unaffected in these Runx1 mutants. These findings appear to argue against the recently proposed role of VGLUT3 in C-LTMRs in mediating mechanical hypersensitivity induced by nerve injury or inflammation. Thus, our studies provide new insight into the genetic program controlling C-LTMR development and call for a revisit for the physiological functions of C-LTMRs.

  11. Indoleamine 2,3-dioxygenase expression in patients with allergic rhinitis: a case-control study

    Directory of Open Access Journals (Sweden)

    Luukkainen Annika

    2011-12-01

    Full Text Available Abstract Background Indoleamine 2,3-dioxygenase (IDO is a tryptophan catalyzing enzyme. It has been suggested that it has a role in lower airway allergic inflammations, but its role in allergic rhinitis has not been investigated. Objective Our aim was to evaluate the expression of IDO in the nasal mucosa of allergic rhinitis patients allergic to birch pollen during peak exposure to birch pollen allergen and compare it to non-atopic patients. Methods IDO expression was immunohistochemically evaluated from nasal specimens obtained in- and off-season from otherwise healthy non-smoking volunteers both allergic to birch pollen (having mild or moderate allergic rhinoconjunctivitis and non-allergic controls. Results: The IDO expression levels were low in healthy controls and remained low also in patients allergic to birch pollen. There were no differences in the expression of IDO in- and off-season in either healthy or allergic subjects. Conclusions There is a controversy in the role of IDO in upper and lower airways during allergic airway disease. It seems that IDO is associated to allergic inflammations of the lower airways, but does not have a local role in the nasal cavity at least in mild or moderate forms of allergic rhinitis.

  12. Polycomb controls gliogenesis by regulating the transient expression of the Gcm/Glide fate determinant.

    Directory of Open Access Journals (Sweden)

    Anna Popkova

    Full Text Available The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system.

  13. CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

    Science.gov (United States)

    Borovsky, Dov; Sterner, Andeas; Powell, Charles A

    2016-01-01

    The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh.

  14. Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

    DEFF Research Database (Denmark)

    Pinto, Rita; Hansen, Lars; Hintze, John Birger Hjalmar

    2017-01-01

    Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven...... to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii......) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide...

  15. The chromatin remodeling factor CHD7 controls cerebellar development by regulating reelin expression

    Science.gov (United States)

    Whittaker, Danielle E.; Riegman, Kimberley L.H.; Kasah, Sahrunizam; Mohan, Conor; Yu, Tian; Sala, Blanca Pijuan; Hebaishi, Husam; Caruso, Angela; Marques, Ana Claudia; Michetti, Caterina; Smachetti, María Eugenia Sanz; Shah, Apar; Sabbioni, Mara; Kulhanci, Omer; Tee, Wee-Wei; Reinberg, Danny; Scattoni, Maria Luisa; McGonnell, Imelda; Wardle, Fiona C.; Fernandes, Cathy

    2017-01-01

    The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of Chd7 from cerebellar granule cell progenitors (GCps) results in reduced GCp proliferation, cerebellar hypoplasia, developmental delay, and motor deficits in mice. Genome-wide expression profiling revealed downregulated expression of the gene encoding the glycoprotein reelin (Reln) in Chd7-deficient GCps. Recessive RELN mutations have been associated with severe cerebellar hypoplasia in humans. We found molecular and genetic evidence that reductions in Reln expression contribute to GCp proliferative defects and cerebellar hypoplasia in GCp-specific Chd7 mouse mutants. Finally, we showed that CHD7 is necessary for maintaining an open, accessible chromatin state at the Reln locus. Taken together, this study shows that Reln gene expression is regulated by chromatin remodeling, identifies CHD7 as a previously unrecognized upstream regulator of Reln, and provides direct in vivo evidence that a mammalian CHD protein can control brain development by modulating chromatin accessibility in neuronal progenitors. PMID:28165338

  16. Direct control of Na(+)-K(+)-2Cl(-)-cotransport protein (NKCC1) expression with aldosterone.

    Science.gov (United States)

    Ding, Bo; Frisina, Robert D; Zhu, Xiaoxia; Sakai, Yoshihisa; Sokolowski, Bernd; Walton, Joseph P

    2014-01-01

    Sodium/potassium/chloride cotransporter (NKCC1) proteins play important roles in Na(+) and K(+) concentrations in key physiological systems, including cardiac, vascular, renal, nervous, and sensory systems. NKCC1 levels and functionality are altered in certain disease states, and tend to decline with age. A sensitive, effective way of regulating NKCC1 protein expression has significant biotherapeutic possibilities. The purpose of the present investigation was to determine if the naturally occurring hormone aldosterone (ALD) could regulate NKCC1 protein expression. Application of ALD to a human cell line (HT-29) revealed that ALD can regulate NKCC1 protein expression, quite sensitively and rapidly, independent of mRNA expression changes. Utilization of a specific inhibitor of mineralocorticoid receptors, eplerenone, implicated these receptors as part of the ALD mechanism of action. Further experiments with cycloheximide (protein synthesis inhibitor) and MG132 (proteasome inhibitor) revealed that ALD can upregulate NKCC1 by increasing protein stability, i.e., reducing ubiquitination of NKCC1. Having a procedure for controlling NKCC1 protein expression opens the doors for therapeutic interventions for diseases involving the mis-regulation or depletion of NKCC1 proteins, for example during aging.

  17. RabGDI controls axonal midline crossing by regulating Robo1 surface expression

    Directory of Open Access Journals (Sweden)

    Philipp Melanie

    2012-11-01

    Full Text Available Abstract Background Axons navigate to their future synaptic targets with the help of choice points, intermediate targets that express axon guidance cues. Once they reach a choice point, axons need to switch their response from attraction to repulsion in order to move on with the next stage of their journey. The mechanisms underlying the change in axonal responsiveness are poorly understood. Commissural axons become sensitive to the repulsive activity of Slits when they cross the ventral midline of the CNS. Responsiveness to Slits depends on surface expression of Robo receptors. In Drosophila, Commissureless (Comm plays a crucial regulatory role in midline crossing by keeping Robo levels low on precommissural axons. Interestingly, to date no vertebrate homolog of comm has been identified. Robo3/Rig1 has been shown to control Slit sensitivity before the midline, but without affecting Robo1 surface expression. Results We had identified RabGDI, a gene linked to human mental retardation and an essential component of the vesicle fusion machinery, in a screen for differentially expressed floor-plate genes. Downregulation of RabGDI by in ovo RNAi caused commissural axons to stall in the floor plate, phenocopying the effect observed after downregulation of Robo1. Conversely, premature expression of RabGDI prevented commissural axons from entering the floor plate. Furthermore, RabGDI triggered Robo1 surface expression in cultured commissural neurons. Taken together, our results identify RabGDI as a component of the switching mechanism that is required for commissural axons to change their response from attraction to repulsion at the intermediate target. Conclusion RabGDI takes over the functional role of fly Comm by regulating the surface expression of Robo1 on commissural axons in vertebrates. This in turn allows commissural axons to switch from attraction to repulsion at the midline of the spinal cord.

  18. Mitogen-activated protein kinase signaling controls basal and oncostatin M-mediated JUNB gene expression.

    Science.gov (United States)

    Hicks, Mellissa J; Hu, Qiuping; Macrae, Erin; DeWille, James

    2015-05-01

    The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

  19. Cultural differences in the links between parental control and children's emotional expressivity.

    Science.gov (United States)

    Louie, Jennifer Y; Oh, Brian J; Lau, Anna S

    2013-10-01

    Research suggests that parental control may be motivated by various socialization goals and contributes to children's adjustment in diverse ways depending on cultural context. The present study examined whether parental psychological control was differentially related to children's emotional expressivity in a sample of 127 Korean, Asian American (AA), and European American (EA) preschoolers. Results indicated that Korean and AA parents endorsed more parental control (emotion suppression, shaming) than EA parents. Similarly, Korean and AA children displayed less observable sadness and exuberance during emotion-eliciting tasks than EA children. Furthermore, moderation analyses revealed that for EA families, parental control was positively correlated with child anger and exuberance; however, the associations were not significant for AA and Korean families. PsycINFO Database Record (c) 2013 APA, all rights reserved.

  20. Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

    Directory of Open Access Journals (Sweden)

    Jessica L Soyer

    2014-03-01

    Full Text Available Plant pathogens secrete an arsenal of small secreted proteins (SSPs acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus. Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3, in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin

  1. Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites.

    Science.gov (United States)

    Merulla, Davide; van der Meer, Jan Roelof

    2016-01-15

    Expression control in synthetic genetic circuitry, for example, for construction of sensitive biosensors, is hampered by the lack of DNA parts that maintain ultralow background yet achieve high output upon signal integration by the cells. Here, we demonstrate how placement of auxiliary transcription factor binding sites within a regulatable promoter context can yield an important gain in signal-to-noise output ratios from prokaryotic biosensor circuits. As a proof of principle, we use the arsenite-responsive ArsR repressor protein from Escherichia coli and its cognate operator. Additional ArsR operators placed downstream of its target promoter can act as a transcription roadblock in a distance-dependent manner and reduce background expression of downstream-placed reporter genes. We show that the transcription roadblock functions both in cognate and heterologous promoter contexts. Secondary ArsR operators placed upstream of their promoter can also improve signal-to-noise output while maintaining effector dependency. Importantly, background control can be released through the addition of micromolar concentrations of arsenite. The ArsR-operator system thus provides a flexible system for additional gene expression control, which, given the extreme sensitivity to micrograms per liter effector concentrations, could be applicable in more general contexts.

  2. Decreased profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.

    Science.gov (United States)

    Nirunsuksiri, W; Presland, R B; Brumbaugh, S G; Dale, B A; Fleckman, P

    1995-01-13

    Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized by mild hyperkeratosis and reduced or absent keratohyalin granules in the epidermis. Profilaggrin, a major component of keratohyalin granules, is reduced or absent from the skin of individuals with ichthyosis vulgaris. In this report, we have further characterized the molecular basis of low profilaggrin expression, which occurs in this disease. In situ hybridization revealed little profilaggrin mRNA in ichthyosis vulgaris-affected epidermis. In keratinocytes cultured from the epidermis of affected individuals, the abundance of profilaggrin was reduced to less than 10% of normal controls, while the mRNA level was decreased to 30-60% of controls. Expression of K1 and loricrin, other markers of epidermal differentiation, were not affected. Nuclear run-on assays indicated that the decrease in mRNA levels was not caused by aberrant transcription. Nucleotide sequencing of 5'-upstream, 3'-non-coding, and flanking regions of the profilaggrin gene from ichthyosis vulgaris-affected individuals revealed only minor changes, probably due to genetic polymorphisms. Our results indicate that defective profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.

  3. Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.

    Science.gov (United States)

    Ueki, Toshiyuki; Lovley, Derek R

    2010-11-01

    Geobacter species often play an important role in bioremediation of environments contaminated with metals or organics and show promise for harvesting electricity from waste organic matter in microbial fuel cells. The ability of Geobacter species to fix atmospheric nitrogen is an important metabolic feature for these applications. We identified novel regulatory cascades controlling nitrogen-fixation gene expression in Geobacter sulfurreducens. Unlike the regulatory mechanisms known in other nitrogen-fixing microorganisms, nitrogen-fixation gene regulation in G. sulfurreducens is controlled by two two-component His-Asp phosphorelay systems. One of these systems appears to be the master regulatory system that activates transcription of the majority of nitrogen-fixation genes and represses a gene encoding glutamate dehydrogenase during nitrogen fixation. The other system whose expression is directly activated by the master regulatory system appears to control by antitermination the expression of a subset of the nitrogen-fixation genes whose transcription is activated by the master regulatory system and whose promoter contains transcription termination signals. This study provides a new paradigm for nitrogen-fixation gene regulation.

  4. NOF1 encodes an Arabidopsis protein involved in the control of rRNA expression.

    Directory of Open Access Journals (Sweden)

    Erwana Harscoët

    Full Text Available The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes.

  5. Bootstrapping of gene-expression data improves and controls the false discovery rate of differentially expressed genes

    Directory of Open Access Journals (Sweden)

    Goddard Mike E

    2004-03-01

    Full Text Available Abstract The ordinary-, penalized-, and bootstrap t-test, least squares and best linear unbiased prediction were compared for their false discovery rates (FDR, i.e. the fraction of falsely discovered genes, which was empirically estimated in a duplicate of the data set. The bootstrap-t-test yielded up to 80% lower FDRs than the alternative statistics, and its FDR was always as good as or better than any of the alternatives. Generally, the predicted FDR from the bootstrapped P-values agreed well with their empirical estimates, except when the number of mRNA samples is smaller than 16. In a cancer data set, the bootstrap-t-test discovered 200 differentially regulated genes at a FDR of 2.6%, and in a knock-out gene expression experiment 10 genes were discovered at a FDR of 3.2%. It is argued that, in the case of microarray data, control of the FDR takes sufficient account of the multiple testing, whilst being less stringent than Bonferoni-type multiple testing corrections. Extensions of the bootstrap simulations to more complicated test-statistics are discussed.

  6. Gene expression profile suggests that pigs (Sus scrofa are susceptible to Anaplasma phagocytophilum but control infection

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    Galindo Ruth C

    2012-08-01

    Full Text Available Abstract Background Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. Results For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1, T-cell receptor alpha chain (TCR-alpha, thrombospondin 4 (TSP-4 and Gap junction protein alpha 1 (GJA1 genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. Conclusions These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

  7. Seasonal changes in patterns of gene expression in avian song control brain regions.

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    Christopher K Thompson

    Full Text Available Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2 0.585 in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.

  8. Drosophila p53 controls Notch expression and balances apoptosis and proliferation.

    Science.gov (United States)

    Simón, Rocío; Aparicio, Ricardo; Housden, Ben E; Bray, Sarah; Busturia, Ana

    2014-10-01

    A balance between cell proliferation and apoptosis is important for normal development and tissue homeostasis. Under stress conditions, the conserved tumor suppressor and transcription factor Dp53 induces apoptosis to contribute to the maintenance of homeostasis. However, in some cases Dp53-induced apoptosis results in the proliferation of surrounding non-apoptotic cells. To gain insight into the Dp53 function in the control of apoptosis and proliferation, we studied the interaction between the Drosophila Dp53 and Notch genes. We present evidence that simultaneous reduction of Dp53 and Notch function synergistically increases the wing phenotype of Notch heterozygous mutant flies. Further, we found that a Notch cis-regulatory element is responsive to loss and gain of Dp53 function and that over-expression of Dp53 up-regulates Notch mRNA and protein expression. These findings suggest not only that Dp53 and Notch act together to control wing development but also indicate that Dp53 transcriptionally regulates Notch expression. Moreover, using Notch  gain and loss of function mutations we examined the relevance of Dp53 and Notch interactions in the process of Dp53-apoptosis induced proliferation. Results show that proliferation induced by Dp53 over-expression is dependent on Notch, thus identifying Notch as a new player in Dp53-induced proliferation. Interestingly, we found that Dp53-induced Notch activation and proliferation occurs even under conditions where apoptosis was inhibited. Our findings highlight the conservation between flies and vertebrates of the Dp53 and Notch cross-talk and suggest that Dp53 has a dual role regulating cell death and proliferation gene networks to control the homeostatic balance between apoptosis and proliferation.

  9. Pyk2 controls filamentous actin formation in human glomerular mesangial cells via modulation of profilin expression

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    Victoriya A Rufanova

    2009-06-01

    Full Text Available Victoriya A Rufanova1, Anna Alexanian1, Tetsuro Wakatsuki2,3, Andrey Sorokin11Department of Medicine, Division of Nephrology, Kidney Disease Center Milwaukee, WI, USA; 2Department of Physiology, 3Bioengineering and Biotechnology Center, Medical College of Wisconsin, Milwaukee, WI, USAAbstract: In glomerular mesangial cell (GMC, important regulators of glomerular filtration, adenovirus-mediated overexpression of calcium regulated nonkinase (CRNK, a dominant interfering calcium-regulated nonreceptor proline-rich tyrosine kinase 2 (Pyk2 construct, inhibited Pyk2 activity and caused enhanced RhoA activity, enriched cortical actin formation at time of cell replating, and reduction of spreading. We aimed to further explore Pyk2 regulation of the actin dynamic during cell spreading as a vital characteristic of GMC function. GMC were infected with adenovirus encoding CRNK or green fluorescent protein (GFP as a control and 48 hours after infection cells were harvested and either re-plated or left in suspension for one hour. De novo adhesion to substrate was significantly decreased after Pyk2 activity inhibition and was further diminished after treatment with Rho-associated kinase inhibitor. Inhibition of Pyk2 was associated with increased filamentous actin formation and a corresponding decrease in globular to filamentous actin ratio during cell spreading. Phosphorylation and expression of cofilin, a RhoA-regulated filamentous actin destabilizing factor, were similar in CRNK-expressing and control GMC. Expression of profilin, an activator of actin polymerization, was enhanced, whereas phosphorylation of Pyk2 and p130Cas was decreased. Our data suggest that Pyk2 signaling controls the filamentous actin formation during cell spreading via upregulation of profilin expression.Keywords: Pyk2, profilin, cell spreading, adhesion, glomerular mesangial cells, p130Cas, actin dynamic, ROCK inhibition

  10. RAE-1 is expressed in the adult subventricular zone and controls cell proliferation of neurospheres.

    Science.gov (United States)

    Popa, Natalia; Cedile, Oriane; Pollet-Villard, Xavier; Bagnis, Claude; Durbec, Pascale; Boucraut, José

    2011-01-01

    Improving and controlling the capacity of endogenous or grafted adult neural stem cells to repair the nervous system relies on a better knowledge of interactions between immune cells and neural stem cells. Class I major histocompatibility complex (MHC) family members comprise numerous proteins playing either immune or nonimmune function. Among the latter, MHC functions in the central nervous system has started to receive recent interest. Here, our first goal was to investigate the potential relationship between MHC class I molecules and neurogenesis. For the first time, we report the expression of two MHC class I-related members by neural stem/progenitor cells: retinoic acid early induced transcript (RAE)-1 and CD1d. The expression of RAE-1 but not CD1d disappears when differentiation of neurosphere cells is induced. Interestingly, RAE-1 transcripts are expressed in the brain during development, and we demonstrate they persist in one of the main area of adult neurogenesis, the subventricular zone (SVZ). So far, RAE-1 is only known for its immune functions as a ligand of the activating receptor NKG2D expressed by natural killer (NK) cells, natural killer T, Tγδ, and some T CD8 lymphocytes. Here, we do not detect any NKG2D expression in the SVZ either in physiological or in pathological conditions. Interestingly, inhibition of RAE-1 expression in neurosphere cells reduces cell proliferation without alteration of cell viability, which argues for a nonimmune role for RAE-1. These results reveal an unexpected role of RAE-1 in regulating adult SVZ neurogenesis by supporting stem/progenitor cells proliferation. © 2010 Wiley-Liss, Inc.

  11. Gene expression profiling in persons with multiple chemical sensitivity before and after a controlled n-butanol exposure session

    Science.gov (United States)

    Dantoft, Thomas M; Skovbjerg, Sine; Andersson, Linus; Claeson, Anna-Sara; Engkilde, Kaare; Lind, Nina; Nordin, Steven; Hellgren, Lars I

    2017-01-01

    Objectives To investigate the pathophysiological pathways leading to symptoms elicitation in multiple chemical sensitivity (MCS) by comparing gene expression in MCS participants and healthy controls before and after a chemical exposure optimised to cause symptoms among MCS participants. The first hypothesis was that unexposed and symptom-free MCS participants have similar gene expression patterns to controls and a second hypothesis that MCS participants can be separated from controls based on differential gene expression upon a controlled n-butanol exposure. Design Participants were exposed to 3.7 ppm n-butanol while seated in a windowed exposure chamber for 60 min. A total of 26 genes involved in biochemical pathways found in the literature have been proposed to play a role in the pathogenesis of MCS and other functional somatic syndromes were selected. Expression levels were compared between MCS and controls before, within 15 min after being exposed to and 4 hours after the exposure. Settings Participants suffering from MCS and healthy controls were recruited through advertisement at public places and in a local newspaper. Participants 36 participants who considered themselves sensitive were prescreened for eligibility. 18 sensitive persons fulfilling the criteria for MCS were enrolled together with 18 healthy controls. Outcome measures 17 genes showed sufficient transcriptional level for analysis. Group comparisons were conducted for each gene at the 3 times points and for the computed area under the curve (AUC) expression levels. Results MCS participants and controls displayed similar gene expression levels both at baseline and after the exposure and the computed AUC values were likewise comparable between the 2 groups. The intragroup variation in expression levels among MCS participants was noticeably greater than the controls. Conclusions MCS participants and controls have similar gene expression levels at baseline and it was not possible to separate

  12. Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes

    Science.gov (United States)

    Lui, Leong T.; Xue, Xuan; Sui, Cheng; Brown, Alan; Pritchard, David I.; Halliday, Nigel; Winzer, Klaus; Howdle, Steven M.; Fernandez-Trillo, Francisco; Krasnogor, Natalio; Alexander, Cameron

    2013-12-01

    Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one method by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric ‘bacteria sequestrants’, designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterize the feedback between bacteria clustering and quorum sensing signalling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population level.

  13. Bacteria clustering by polymers induces the expression of quorum-sensing-controlled phenotypes.

    Science.gov (United States)

    Lui, Leong T; Xue, Xuan; Sui, Cheng; Brown, Alan; Pritchard, David I; Halliday, Nigel; Winzer, Klaus; Howdle, Steven M; Fernandez-Trillo, Francisco; Krasnogor, Natalio; Alexander, Cameron

    2013-12-01

    Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one method by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric 'bacteria sequestrants', designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterize the feedback between bacteria clustering and quorum sensing signalling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population level.

  14. Cohesins: chromatin architects in chromosome segregation, control of gene expression and much more.

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    Barbero, José L

    2009-07-01

    Cells have evolved to develop molecules and control mechanisms that guarantee correct chromosome segregation and ensure the proper distribution of genetic material to daughter cells. In this sense, the establishment, maintenance, and removal of sister chromatid cohesion is one of the most fascinating and dangerous processes in the life of a cell because errors in the control of these processes frequently lead to cell death or aneuploidy. The main protagonist in this mechanism is a four-protein complex denominated the cohesin complex. In the last 10 years, we have improved our understanding of the key players in the regulation of sister chromatid cohesion during cell division in mitosis and meiosis. The last 2 years have seen an increase in evidence showing that cohesins have important functions in non-dividing cells, revealing new, unexplored roles for these proteins in the control of gene expression, development, and other essential cell functions in mammals.

  15. The expression of stlA in Photorhabdus luminescens is controlled by nutrient limitation.

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    Lea Lango-Scholey

    Full Text Available Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σ(S and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism.

  16. Regulatory elements controlling pituitary-specific expression of the human prolactin gene.

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    Peers, B; Voz, M L; Monget, P; Mathy-Hartert, M; Berwaer, M; Belayew, A; Martial, J A

    1990-09-01

    We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

  17. Expression of Arabidopsis hexokinase in citrus guard cells controls stomatal aperture and reduces transpiration

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    Nitsan eLugassi

    2015-12-01

    Full Text Available Hexokinase (HXK is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1 under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

  18. ABA-mediated ROS in mitochondria regulate root meristem activity by controlling PLETHORA expression in Arabidopsis.

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    Li Yang

    2014-12-01

    Full Text Available Although research has determined that reactive oxygen species (ROS function as signaling molecules in plant development, the molecular mechanism by which ROS regulate plant growth is not well known. An aba overly sensitive mutant, abo8-1, which is defective in a pentatricopeptide repeat (PPR protein responsible for the splicing of NAD4 intron 3 in mitochondrial complex I, accumulates more ROS in root tips than the wild type, and the ROS accumulation is further enhanced by ABA treatment. The ABO8 mutation reduces root meristem activity, which can be enhanced by ABA treatment and reversibly recovered by addition of certain concentrations of the reducing agent GSH. As indicated by low ProDR5:GUS expression, auxin accumulation/signaling was reduced in abo8-1. We also found that ABA inhibits the expression of PLETHORA1 (PLT1 and PLT2, and that root growth is more sensitive to ABA in the plt1 and plt2 mutants than in the wild type. The expression of PLT1 and PLT2 is significantly reduced in the abo8-1 mutant. Overexpression of PLT2 in an inducible system can largely rescue root apical meristem (RAM-defective phenotype of abo8-1 with and without ABA treatment. These results suggest that ABA-promoted ROS in the mitochondria of root tips are important retrograde signals that regulate root meristem activity by controlling auxin accumulation/signaling and PLT expression in Arabidopsis.

  19. Robust control of the seasonal expression of the Arabidopsis FLC gene in a fluctuating environment.

    Science.gov (United States)

    Aikawa, Shinichiro; Kobayashi, Masaki J; Satake, Akiko; Shimizu, Kentaro K; Kudoh, Hiroshi

    2010-06-22

    Plants flower in particular seasons even in natural, fluctuating environments. The molecular basis of temperature-dependent flowering-time regulation has been extensively studied, but little is known about how gene expression is controlled in natural environments. Without a memory of past temperatures, it would be difficult for plants to detect seasons in natural, noisy environments because temperature changes occurring within a few weeks are often inconsistent with seasonal trends. Our 2-y census of the expression of a temperature-dependent flowering-time gene, AhgFLC, in a natural population of perennial Arabidopsis halleri revealed that the regulatory system of this flowering-time gene extracts seasonal cues as if it memorizes temperatures over the past 6 wk. Time-series analysis revealed that as much as 83% of the variation in the AhgFLC expression is explained solely by the temperature for the previous 6 wk, but not by the temperatures over shorter or longer periods. The accuracy of our model in predicting the gene expression pattern under contrasting temperature regimes in the transplant experiments indicates that such modeling incorporating the molecular bases of flowering-time regulation will contribute to predicting plant responses to future climate changes.

  20. Hoxb8 regulates expression of microRNAs to control cell death and differentiation.

    Science.gov (United States)

    Salmanidis, M; Brumatti, G; Narayan, N; Green, B D; van den Bergen, J A; Sandow, J J; Bert, A G; Silke, N; Sladic, R; Puthalakath, H; Rohrbeck, L; Okamoto, T; Bouillet, P; Herold, M J; Goodall, G J; Jabbour, A M; Ekert, P G

    2013-10-01

    Hoxb8 overexpression immortalises haematopoietic progenitor cells in a growth-factor-dependant manner and co-operates with interleukin-3 (IL-3) to cause acute myeloid leukaemia. To further understand how Hoxb8 contributes to myeloid cell immortalisation, we generated IL-3-dependant myeloid cells expressing Hoxb8 under the control of an inducible promoter. Downregulation of Hoxb8, in the presence of IL-3, caused cell-cycle arrest and apoptosis in the majority of cells. Apoptosis was dependant on Bax and Bak and, in part, on Bim, which was repressed by Hoxb8. Deletion of the miR-17∼92 seed sequences in the Bim 3'UTR abolished Hoxb8-dependant regulation of Bim reporter constructs. Expression of all six miRNAs from this cluster were elevated when Hoxb8 was overexpressed. The miR-17∼92 cluster was required for repression of Bim in Hoxb8-immortalised cells and deletion of the miR-17∼92 cluster substantially inhibited Hoxb8, but not Hoxa9, mediated survival and proliferation. Hoxb8 appears to promote miR-17∼92 expression through c-Myc, a known transcriptional regulator of the miR-17∼92 cluster. We have uncovered a previously unrecognised link between Hoxb8 expression and microRNAs that provides a new insight into the oncogenic functions of Hoxb8.

  1. Cryptic Transcription and Early Termination in the Control of Gene Expression

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    Jessie Colin

    2011-01-01

    Full Text Available Recent studies on yeast transcriptome have revealed the presence of a large set of RNA polymerase II transcripts mapping to intergenic and antisense regions or overlapping canonical genes. Most of these ncRNAs (ncRNAs are subject to termination by the Nrd1-dependent pathway and rapid degradation by the nuclear exosome and have been dubbed cryptic unstable transcripts (CUTs. CUTs are often considered as by-products of transcriptional noise, but in an increasing number of cases they play a central role in the control of gene expression. Regulatory mechanisms involving expression of a CUT are diverse and include attenuation, transcriptional interference, and alternative transcription start site choice. This review focuses on the impact of cryptic transcription on gene expression, describes the role of the Nrd1-complex as the main actor in preventing nonfunctional and potentially harmful transcription, and details a few systems where expression of a CUT has an essential regulatory function. We also summarize the most recent studies concerning other types of ncRNAs and their possible role in regulation.

  2. THE EXPRESSION AND CLINICAL VALUE OF APOPTOSIS CONTROL GENE Bcl-2 AND Bax IN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jun; YAO Zhen-xiang; ZHANG Jing

    1999-01-01

    Objective: To study the expression and clinical value of apoptosis control gene bcl-2 and bax in breast cancer.Methods: Protein bax and bcl-2 in 41 breast cancers obtained from operations in our hospital in 1996 were detected using ABC immunohistochemical stain assay and compared with 10 cases with normal breast tissues.Results: The positive rate of bax in normal breast tissue was 90% and in breast cancer was 59%, with a significant statistical difference between them (P<0.05), but there was no statistical difference in bcl-2 protein expression. Among the 41 breast cancer, the group with lymph node metastasis (21 cases) had obviously low bax expression (43%) and high bcl-2 expression (76%), showing significant difference to the group without lymph node metastasis (P<0.05).Conclusion: The antiapoptosis function of bcl-2 was stronger than bax in breast cancer. Protein bax and bcl-2 assay may be useful in understanding the biological behaviors of breast cancer.

  3. ABA-mediated ROS in mitochondria regulate root meristem activity by controlling PLETHORA expression in Arabidopsis.

    Science.gov (United States)

    Yang, Li; Zhang, Jing; He, Junna; Qin, Yingying; Hua, Deping; Duan, Ying; Chen, Zhizhong; Gong, Zhizhong

    2014-12-01

    Although research has determined that reactive oxygen species (ROS) function as signaling molecules in plant development, the molecular mechanism by which ROS regulate plant growth is not well known. An aba overly sensitive mutant, abo8-1, which is defective in a pentatricopeptide repeat (PPR) protein responsible for the splicing of NAD4 intron 3 in mitochondrial complex I, accumulates more ROS in root tips than the wild type, and the ROS accumulation is further enhanced by ABA treatment. The ABO8 mutation reduces root meristem activity, which can be enhanced by ABA treatment and reversibly recovered by addition of certain concentrations of the reducing agent GSH. As indicated by low ProDR5:GUS expression, auxin accumulation/signaling was reduced in abo8-1. We also found that ABA inhibits the expression of PLETHORA1 (PLT1) and PLT2, and that root growth is more sensitive to ABA in the plt1 and plt2 mutants than in the wild type. The expression of PLT1 and PLT2 is significantly reduced in the abo8-1 mutant. Overexpression of PLT2 in an inducible system can largely rescue root apical meristem (RAM)-defective phenotype of abo8-1 with and without ABA treatment. These results suggest that ABA-promoted ROS in the mitochondria of root tips are important retrograde signals that regulate root meristem activity by controlling auxin accumulation/signaling and PLT expression in Arabidopsis.

  4. NFI-C regulates osteoblast differentiation via control of osterix expression.

    Science.gov (United States)

    Lee, Dong-Seol; Choung, Han-Wool; Kim, Heung-Joong; Gronostajski, Richard M; Yang, Young-Il; Ryoo, Hyun-Mo; Lee, Zang Hee; Kim, Hong-Hee; Cho, Eui-Sic; Park, Joo-Cheol

    2014-09-01

    In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age-related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I-C (NFI-C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI-C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI-C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic-overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator-activated receptor gamma expression in Nfic(-/-) mice showing an age-related osteoporosis-like phenotype. Finally, NFI-C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein-2-Runx2 pathway. These results suggest that NFI-C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI-C expression in BMSCs could be a novel therapeutic approach for treating age-related osteoporosis.

  5. A new role for GABAergic transmission in the control of male rat sexual behavior expression.

    Science.gov (United States)

    Rodríguez-Manzo, Gabriela; Canseco-Alba, Ana

    2017-03-01

    GABAergic transmission in the ventral tegmental area (VTA) exerts a tonic inhibitory influence on mesolimbic dopaminergic neurons' activity. Blockade of VTA GABAA receptors increases dopamine release in the nucleus accumbens (NAcc). Increases in NAcc dopamine levels typically accompany sexual behavior display. Copulation to satiety is characterized by the instatement of a long lasting (72h) sexual behavior inhibition and the mesolimbic system appears to be involved in this phenomenon. GABAergic transmission in the VTA might play a role in the maintenance of this long lasting sexual inhibitory state. To test this hypothesis, in the present work we investigated the effect of GABAA receptor blockade in sexually exhausted males 24h after copulation to satiety, once the sexual inhibitory state is established, and compared it with its effect in sexually experienced rats. Results showed that low doses of systemically administered bicuculline induced sexual behavior expression in sexually exhausted rats, but lacked an effect on copulation of sexually experienced animals. Intra-VTA bilateral infusion of bicuculline did not modify sexual behavior of sexually experienced rats, but induced sexual behavior expression in all the sexually exhausted males. Hence, GABA plays a role in the control of sexual behavior expression at the VTA. The role played by GABAergic transmission in male sexual behavior expression of animals with distinct sexual behavior conditions is discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Expression liver-directed genes by employing synthetic transcriptional control units

    Institute of Scientific and Technical Information of China (English)

    Marie-Luise Lemken; Wolfgang A. Wybranietz; Ulrike Schmidt; Florian Graepler; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer

    2005-01-01

    AIM: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver,thereby compensating for the lack of specificity of currently available gene therapeutic vector systems.METHODS: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neor fusion gene cassette was generated,and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis.CONCLUSION: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver

  7. Cosmetics-triggered percutaneous remote control of transgene expression in mice

    Science.gov (United States)

    Wang, Hui; Ye, Haifeng; Xie, Mingqi; Daoud El-Baba, Marie; Fussenegger, Martin

    2015-01-01

    Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies. PMID:25943548

  8. Electrotransfer parameters as a tool for controlled and targeted gene expression in skin

    Directory of Open Access Journals (Sweden)

    Spela Kos

    2016-01-01

    Full Text Available Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed. Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

  9. Cosmetics-triggered percutaneous remote control of transgene expression in mice.

    Science.gov (United States)

    Wang, Hui; Ye, Haifeng; Xie, Mingqi; Daoud El-Baba, Marie; Fussenegger, Martin

    2015-08-18

    Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies.

  10. Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

    Science.gov (United States)

    Zhong, Guocai; Wang, Haimin; Bailey, Charles C; Gao, Guangping; Farzan, Michael

    2016-01-01

    Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes’s activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules. DOI: http://dx.doi.org/10.7554/eLife.18858.001 PMID:27805569

  11. Cloning, expression and characterization of translationally controlled tumor protein (TCTP) gene from flatfish turbot ( Scophthalmus maximus)

    Science.gov (United States)

    Wang, Jian; Guo, Huarong; Zhang, Shicui; Yin, Licheng; Guo, Bin; Wang, Shaojie

    2008-05-01

    A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot ( Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading frame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known vertebrate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCP of turbot and sea perch ( Lateolabrax japonicus) is 170 aa in length, while that of zebrafish ( Danio rerio) and rohu ( Labeo rohita) is 171 aa in length. Northern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET30a- SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further investigation of the biological function of TCTP in fish.

  12. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  13. The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

    Directory of Open Access Journals (Sweden)

    Kyle A. Nilson

    2011-01-01

    Full Text Available HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II, leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

  14. Matrix expression and vaccination control for epidemic dynamics over dynamic networks

    Institute of Scientific and Technical Information of China (English)

    Peilian GUO; Yuzhen WANG

    2016-01-01

    This paper investigates epidemic dynamics over dynamic networks via the approach of semi-tensor product of matrices. First, a formal susceptible-infected-susceptible epidemic dynamic model over dynamic networks (SISED-DN) is given. Second, based on a class of determinate co-evolutionary rule, the matrix expressions are established for the dynamics of individual states and network topologies, respectively. Then, all possible final spreading equilibria are obtained for any given initial epidemic state and network topology by the matrix expression. Third, a sufficient and necessary condition of the existence of state feedback vaccination control is presented to make every individual susceptible. The study of illustrative examples shows the effectiveness of our new results.

  15. DEK Expression is controlled by E2F and deregulated in diverse tumor types.

    Science.gov (United States)

    Carro, Maria Stella; Spiga, Fabio Mario; Quarto, Micaela; Di Ninni, Valentina; Volorio, Sara; Alcalay, Myriam; Müller, Heiko

    2006-06-01

    Deregulation of the retinoblastoma (pRB) tumor suppressor pathway associated with aberrant activity of E2F transcription factors is frequently observed in human cancer. Microarray based analyses have revealed a large number of potential downstream mediators of the tumor suppressing activity of pRB, including DEK, a fusion partner of CAN found in a subset of acute myeloid leukaemia (AML) patients carrying a (6; 9) translocation. Here we report that the expression of DEK is under direct control of E2F transcription factors. Chromatin immunoprecipitation assays show that the DEK promoter is bound by endogenous E2F in vivo. The DEK promoter is transactivated by E2F and mutation of E2F binding sites eliminates this effect. Expression levels of DEK in human tumors have been investigated by tissue micro array analysis. We find that DEK is overexpressed in many solid tumors such as colon cancer, larynx cancer, bladder cancer, and melanoma.

  16. α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations.

    Science.gov (United States)

    Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

    2013-02-15

    GABA(A) receptors are critically involved in hippocampal oscillations. GABA(A) receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABA(A) receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABA(A) receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABA(A) receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABA(A) receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations.

  17. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls

    Directory of Open Access Journals (Sweden)

    Noerholm Mikkel

    2012-01-01

    Full Text Available Abstract Background RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients. Methods Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9 and normal controls (N = 7 were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups. Results Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size Conclusions Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size

  18. Computational design of a Zn2+ receptor that controls bacterial gene expression

    Science.gov (United States)

    Dwyer, M. A.; Looger, L. L.; Hellinga, H. W.

    2003-09-01

    The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a -galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.

  19. Validation of endogenous control genes for gene expression studies on human ocular surface epithelium.

    Directory of Open Access Journals (Sweden)

    Bina Kulkarni

    Full Text Available PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG with quantitative reverse transcription PCR (qPCR, for identification of stably expressed endogenous control genes in the ocular surface (OS epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH, beta actin (ACTB, peptidylprolyl isomerase (PPIA, TATA-box binding protein (TBP1, hypoxanthine guanine phosphoribosyl transferase (HPRT1, beta glucuronidase (GUSB, Eucaryotic 18S ribosomal RNA (18S, phosphoglycerate kinase (PGK1, beta-2-microglobulin (B2M, ribosomal protein, large, P0 (RPLP0--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18Sexpressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use

  20. Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells.

    Science.gov (United States)

    Natarajan, Kartiga; Gottipati, Koteswara R; Berhane, Kiflu; Samten, Buka; Pendurthi, Usha; Boggaram, Vijay

    2016-10-22

    Persistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial cells. The effects of dust extracts on inflammatory gene expression were analyzed by quantitative polymerase chain reaction (qPCR), enzyme linked immunosorbent (ELISA) and western blot assays. Oxidant stress was probed by dihydroethidium (DHE) labeling, and immunostaining for 4-hydroxynonenal (4-HNE). Effects on interleukin-8 (IL-8) promoter regulation were determined by transient transfection assay. Dust extracts contained trypsin and elastase activities, and activated protease activated receptor (PAR)-1 and -2. Serine protease inhibitors and PAR-1 or PAR-2 knockdown suppressed inflammatory gene induction. Dust extract induction of IL-8 gene expression was associated with increased DHE-fluorescence and 4-HNE staining, and antioxidants suppressed inflammatory gene induction. Protease inhibitors and antioxidants suppressed protein kinase C and NF-κB activation and induction of IL-8 promoter activity in cells exposed to dust extract. Our studies demonstrate that proteases and intracellular oxidants control organic dust induction of inflammatory gene expression in lung epithelial cells. Targeting proteases and oxidant stress may serve as novel approaches for the treatment of organic dust induced lung diseases. This is the first report on the involvement of oxidant stress in the induction of inflammatory gene expression by organic dust.

  1. Thyroid hormone and adrenergic signaling interact to control pineal expression of the dopamine receptor D4 gene (Drd4)

    DEFF Research Database (Denmark)

    Kim, Jong-So; Bailey, Michael J; Weller, Joan L;

    2009-01-01

    . Our studies indicate that Drd4 is the dominant dopamine receptor gene expressed in the pineal gland. The gene is expressed in pinealocytes at levels which are approximately 100-fold greater than in other tissues, except the retina, in which transcript levels are similar. Pineal Drd4 expression...... and whether thyroid hormone controls expression of other genes in the pineal gland.......Dopamine plays diverse and important roles in vertebrate biology, impacting behavior and physiology through actions mediated by specific G-protein-coupled receptors, one of which is the dopamine receptor D4 (Drd4). Here we present studies on the >100-fold daily rhythm in rat pineal Drd4 expression...

  2. Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications

    Directory of Open Access Journals (Sweden)

    Mond James

    2005-05-01

    Full Text Available Abstract Background The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. Results In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture, the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. Conclusion Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein.

  3. Evaluation of real-time PCR endogenous control genes for analysis of gene expression in bovine endometrium

    Directory of Open Access Journals (Sweden)

    Mitchell Murray D

    2009-11-01

    Full Text Available Abstract Background Quantitative real-time PCR gene expression results are generally normalised using endogenous control genes. These reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. There has been no systematic investigation of endogenous control genes for bovine endometrium to date. The suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. Fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling Holstein-Friesian dairy cows from two divergent genetic backgrounds. Results The expression profiles of five commonly used endogenous control genes (GAPDH, PPIA, RPS9, RPS15A, and UXT and 10 experimentally derived candidate endogenous control genes (SUZ12, C2ORF29, ZNF131, ACTR1A, HDAC1, SLC30A6, CNOT7, DNAJC17, BBS2, and RANBP10 were analysed across 44 samples to determine the most stably expressed gene. Gene stability was assessed using the statistical algorithms GeNorm and Normfinder. All genes presented with low overall variability (0.87 to 1.48% CV of Cq. However, when used to normalise a differentially expressed gene (oxytocin receptor - OXTR in the samples, the reported relative gene expression levels were significantly affected by the control gene chosen. Based on the results of this analysis, SUZ12 is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle. Conclusion This study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates.

  4. Adaptive Representations for Improving Evolvability, Parameter Control, and Parallelization of Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Nigel P. A. Browne

    2010-01-01

    Full Text Available Gene Expression Programming (GEP is a genetic algorithm that evolves linear chromosomes encoding nonlinear (tree-like structures. In the original GEP algorithm, the genome size is problem specific and is determined through trial and error. In this work, a method for adaptive control of the genome size is presented. The approach introduces mutation, transposition, and recombination operators that enable a population of heterogeneously structured chromosomes, something the original GEP algorithm does not support. This permits crossbreeding between normally incompatible individuals, speciation within a population, increases the evolvability of the representations, and enhances parallel GEP. To test our approach, an assortment of problems were used, including symbolic regression, classification, and parameter optimization. Our experimental results show that our approach provides a solution for the problem of self-adaptive control of the genome size of GEP's representation.

  5. YmoA negatively controls the expression of insecticidal genes in Yersinia enterocolitica.

    Science.gov (United States)

    Starke, Mandy; Fuchs, Thilo M

    2014-04-01

    Yersinia enterocolitica is toxic towards invertebrates due to the presence of the toxin complex (tc) genes that are activated by the thermolabile regulator TcaR2. In the search for further regulatory factors involved in insecticidal gene expression, the modulator of yersinial virulence, YmoA, was identified to silence all tc genes of the Y. enterocolitica strain W22703 (biovar 2, serovar O:9). Using promoter fusions with the luciferase reporter, we found that the deletion of ymoA results in elevated transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at both 15 °C and 37 °C. Complementation by episomal ymoA significantly reduced tc gene expression, thus validating the inhibitory activity of YmoA on the production of insecticidal proteins. YmoA contributes to the binding properties of H-NS to the tc promoters by forming a complex with this nucleoid-associated protein, and this complex not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB that play an important role in controlling the expression of both genes. At low temperature, the intracellular amount of thermostable YmoA is not reduced, but the repressor is less functional. These data point to H-NS/YmoA as an antagonist of the inducer TcaR2.

  6. Determination of internal controls for quantitative gene expression of Isochrysis zhangjiangensis at nitrogen stress condition

    Science.gov (United States)

    Wu, Shuang; Zhou, Jiannan; Cao, Xupeng; Xue, Song

    2016-02-01

    Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under nitrogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction (qPCR) provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin (ACT), α-tubulin (TUA), ß-tubulin (TUB), ubiquitin (UBI), 18S rRNA (18S) and one target gene, diacylglycerol acyltransferase (DGAT), were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed I. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experimental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of I. zhangjiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

  7. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

    Science.gov (United States)

    Schmitt, Anja; Grondona, Paula; Maier, Tabea; Brändle, Marc; Schönfeld, Caroline; Jäger, Günter; Kosnopfel, Corinna; Eberle, Franziska C; Schittek, Birgit; Schulze-Osthoff, Klaus; Yazdi, Amir S; Hailfinger, Stephan

    2016-04-01

    The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

  8. Id1 restrains p21 expression to control endothelial progenitor cell formation.

    Directory of Open Access Journals (Sweden)

    Alessia Ciarrocchi

    Full Text Available Loss of Id1 in the bone marrow (BM severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis.

  9. Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals.

    Science.gov (United States)

    Lakowski, Jörn; Majumder, Anirban; Lauderdale, James D

    2007-07-15

    The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.

  10. Phytoestrogens modulate hepcidin expression by Nrf2: Implications for dietary control of iron absorption.

    Science.gov (United States)

    Bayele, Henry K; Balesaria, Sara; Srai, Surjit K S

    2015-12-01

    Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis.

  11. Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression.

    Science.gov (United States)

    Perez, Joëlle; Dansou, Boris; Hervé, Roxane; Levi, Charlène; Tamouza, Houda; Vandermeersch, Sophie; Demey-Thomas, Emmanuelle; Haymann, Jean-Philippe; Zafrani, Lara; Klatzmann, David; Boissier, Marie-Christophe; Letavernier, Emmanuel; Baud, Laurent

    2016-01-01

    Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.

  12. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.

    Science.gov (United States)

    Curran, Kathleen A; Karim, Ashty S; Gupta, Akash; Alper, Hal S

    2013-09-01

    Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between an expression-enhancing terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing an expression-enhancing terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with an expression-enhancing terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.

  13. TRAP1 controls mitochondrial fusion/fission balance through Drp1 and Mff expression.

    Directory of Open Access Journals (Sweden)

    Hironori Takamura

    Full Text Available Mitochondria are dynamic organelles that change in response to extracellular stimuli. These changes are essential for normal mitochondrial/cellular function and are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Although some molecules have been identified to mediate the mitochondrial fusion and fission process, the underlying mechanisms remain unclear. Tumor necrosis factor receptor-associated protein 1 (TRAP1 is a mitochondrial molecule that regulates a variety of mitochondrial functions. Here, we examined the role of TRAP1 in the regulation of morphology. Stable TRAP1 knockdown cells showed abnormal mitochondrial morphology, and we observed significant decreases in dynamin-related protein 1 (Drp1 and mitochondrial fission factor (Mff, mitochondrial fission proteins. Similar results were obtained by transient knockdown of TRAP1 in two different cell lines, SH-SY5Y neuroblastoma cells and KNS-42 glioma cells. However, TRAP1 knockdown did not affect expression levels of fusion proteins. The reduction in Drp1 and Mff protein levels was rescued following treatment with the proteasome inhibitor MG132. These results suggest that TRAP1 regulates the expression of fission proteins and controls mitochondrial fusion/fission, which affects mitochondrial/cellular function.

  14. Production of pigs expressing a transgene under the control of a tetracycline-inducible system.

    Directory of Open Access Journals (Sweden)

    Yong-Xun Jin

    Full Text Available Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP. At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.

  15. Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

    Directory of Open Access Journals (Sweden)

    Jung Hwan Kim

    Full Text Available BACKGROUND: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs, we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages. METHODOLOGY/PRINCIPAL FINDINGS AND CONCLUSIONS: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

  16. Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

    Science.gov (United States)

    Kim, Jung Hwan; Jee, Min Ki; Lee, So Young; Han, Tae Hee; Kim, Bong Sun; Kang, Kyung Sun; Kang, Soo Kyung

    2009-09-24

    To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages. Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

  17. E2F Transcription Factors Control the Roller Coaster Ride of Cell Cycle Gene Expression.

    Science.gov (United States)

    Thurlings, Ingrid; de Bruin, Alain

    2016-01-01

    Initially, the E2F transcription factor was discovered as a factor able to bind the adenovirus E2 promoter and activate viral genes. Afterwards it was shown that E2F also binds to promoters of nonviral genes such as C-MYC and DHFR, which were already known at that time to be important for cell growth and DNA metabolism, respectively. These findings provided the first clues that the E2F transcription factor might be an important regulator of the cell cycle. Since this initial discovery in 1987, several additional E2F family members have been identified, and more than 100 targets genes have been shown to be directly regulated by E2Fs, the majority of these are important for controlling the cell cycle. The progression of a cell through the cell cycle is accompanied with the increased expression of a specific set of genes during one phase of the cell cycle and the decrease of the same set of genes during a later phase of the cell cycle. This roller coaster ride, or oscillation, of gene expression is essential for the proper progression through the cell cycle to allow accurate DNA replication and cell division. The E2F transcription factors have been shown to be critical for the temporal expression of the oscillating cell cycle genes. This review will focus on how the oscillation of E2Fs and their targets is regulated by transcriptional, post-transcriptional and post-translational mechanism in mammals, yeast, flies, and worms. Furthermore, we will discuss the functional impact of E2Fs on the cell cycle progression and outline the consequences when E2F expression is disturbed.

  18. GFI1B controls its own expression binding to multiple sites.

    Science.gov (United States)

    Anguita, Eduardo; Villegas, Ana; Iborra, Francisco; Hernández, Aurora

    2010-01-01

    Transcription factors play essential roles in both normal and malignant hematopoiesis. This is the case for the growth factor independent 1b (GFI1B) transcription factor, which is required for erythroid and megakaryocytic differentiation and over-expressed in leukemic patients and cell lines. To investigate GFI1B regulation, we searched for multispecies conserved non-coding elements between GFI1B and neighboring genes. We used a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay and DNase1 hypersensitivity to assess the chromatin conformation of these sites. Next, we analyzed transcription factor binding and histone modifications at the GFI1B locus including the conserved non-coding elements by a chromatin immunoprecipitation assay. Finally, we studied the interaction of the GFI1B promoter and the conserved non-coding elements with the chromatin conformation capture technique and used immunofluorescence to evaluate GFI1B levels in individual cells. We localized several conserved non-coding elements containing multiple erythroid specific transcription factor binding sites at the GFI1B locus. In GFI1B-expressing cells a subset of these conserved non-coding elements and the promoter adopt a close spatial conformation, localize with open chromatin sites, harbor chromatin modifications associated with gene activation and bind multiple transcription factors and co-repressors. Conclusions Our findings indicate that GFI1B regulatory elements behave as activators and repressors. Different protein levels within a cell population suggest that cells must activate and repress GFI1B continuously to control its final level. These data are consistent with a model of GFI1B regulation in which GFI1B binds to its own promoter and to the conserved non-coding elements as its levels rise. This would attract repressor complexes that progressively down-regulate the gene. GFI1B expression would decrease until a stage at which the activating complexes predominate and

  19. Cloning, Expression and Characterization of Translationally Controlled Tumor Protein (TCTP) Gene from Flatfish Turbot (Scophthalmus maximus)

    Institute of Scientific and Technical Information of China (English)

    WANG Jian; GUO Huarong; ZHANG Shicui; YIN Licheng; GUO Bin; WANG Shaojie

    2008-01-01

    A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish.

  20. Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation.

    Science.gov (United States)

    Ledford, Kelley L; Martinez-De Luna, Reyna I; Theisen, Matthew A; Rawlins, Karisa D; Viczian, Andrea S; Zuber, Michael E

    2017-06-15

    The eye field transcription factor, Six6, is essential for both the early (specification and proliferative growth) phase of eye formation, as well as for normal retinal progenitor cell differentiation. While genomic regions driving six6 optic cup expression have been described, the sequences controlling eye field and optic vesicle expression are unknown. Two evolutionary conserved regions 5' and a third 3' to the six6 coding region were identified, and together they faithfully replicate the endogenous X. laevis six6 expression pattern. Transgenic lines were generated and used to determine the onset and expression patterns controlled by the regulatory regions. The conserved 3' region was necessary and sufficient for eye field and optic vesicle expression. In contrast, the two conserved enhancer regions located 5' of the coding sequence were required together for normal optic cup and mature retinal expression. Gain-of-function experiments indicate endogenous six6 and GFP expression in F1 transgenic embryos are similarly regulated in response to candidate trans-acting factors. Importantly, CRISPR/CAS9-mediated deletion of the 3' eye field/optic vesicle enhancer in X. laevis, resulted in a reduction in optic vesicle size. These results identify the cis-acting regions, demonstrate the modular nature of the elements controlling early versus late retinal expression, and identify potential regulators of six6 expression during the early stages of eye formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Autoimmune regulator (Aire) controls the expression of microRNAs in medullary thymic epithelial cells.

    Science.gov (United States)

    Macedo, Claudia; Evangelista, Adriane F; Marques, Márcia M; Octacílio-Silva, Shirlei; Donadi, Eduardo A; Sakamoto-Hojo, Elza T; Passos, Geraldo A

    2013-04-01

    The autoimmune regulator (Aire) is a transcription factor that controls the ectopic expression of a large set of peripheral tissue antigen (PTA) genes in medullary thymic epithelial cells (mTECs). Recent evidence has demonstrated that Aire releases stalled RNA polymerase II (RNA Pol II) from blockage at the promoter region of its target genes. Given that, in addition to messenger RNAs (mRNA), RNA Pol II also transcribes microRNAs (miRNAs), we raised the hypothesis that Aire might play a role as an upstream controller of miRNA transcription. To test this, we initially analyzed the expression profiles of 662 miRNAs in control and Aire-silenced (siRNA) murine mTEC 3.10 cells using microarrays. The bioinformatics programs SAM and Cluster-TreeView were then used to identify the differentially expressed miRNAs and their profiles, respectively. Thirty Aire-dependent miRNAs were identified in the Aire-silenced mTECs, of which 18 were up- and 12 were down-regulated. The down-regulated miR-376 family was the focus of this study because its members (miR-376a, miR-376b and miR-376c) are located in the genome within the Gm2922 open-reading frame (ORF) gene segment on the chromosome 12F1. The T-boxes (TTATTA) and G-boxes (GATTGG), which represent putative RNA Pol II promoter motifs, were located in a portion spanning 10 kb upstream of the ATG codon of Gm2922. Moreover, we found that Gm2922 encodes an mRNA, which was also down-regulated in Aire-silenced mTECs. These results represent the first evidence that Aire can play a role as a controller of transcription of miRNAs located within genomic regions encompassing ORF and/or mRNA genes. Copyright © 2012 Elsevier GmbH. All rights reserved.

  2. Interleukin-1 controls the constitutive expression of the Cyp7a1 gene by regulating the expression of Cyp7a1 transcriptional regulators in the mouse liver.

    Science.gov (United States)

    Kojima, Misaki; Ashino, Takashi; Yoshida, Takemi; Iwakura, Yoichiro; Degawa, Masakuni

    2011-01-01

    Our previous study using interleukin-1α/β-knockout (IL-1-KO) and wild-type (WT) mice demonstrated that IL-1 acts as a positive factor for constitutive gene expression of hepatic cytochrome P4507a1 (Cyp7a1). In this study, to clarify the role of IL-1 in the expression of the hepatic Cyp7a1 gene, we focused on Cyp7a1 transcriptional regulators such as α-fetoprotein transcription factor (FTF), liver X receptor α (LXRα), hepatocyte nuclear factor 4α (HNF4α) and small heterodimer partner (SHP) and examined the effects of IL-1 on their gene expression by real-time reverse-transcription polymerase chain reaction using IL-1-KO and WT mice. We observed no significant differences between sex-matched IL-1-KO and WT mice with regard to gene expression levels of FTF, LXRα, and HNF4α, all of which are positive transcriptional regulators for the Cyp7a1 gene. However, interindividual differences in hepatic FTF and LXRα expression were closely dependent on the gene expression level(s) of hepatic IL-1 and tumor necrosis factor-α (TNF-α), while interindividual differences in hepatic HNF4α were clearly correlated with the expression of IL-1, but not TNF-α. In contrast, the gene expression level of SHP, which is a negative transcriptional regulator of the Cyp7a1 gene through inhibition of FTF function, was higher in IL-1-KO mice than in sex-matched WT mice. These findings demonstrate that, like TNF-α, IL-1 positively controls the gene expression of Cyp7a1 transcriptional upregulators but, in contrast to the previously reported action of TNF-α, IL-1 also acts to downregulate SHP gene expression.

  3. Neurophysiology of spontaneous facial expressions: I. Motor control of the upper and lower face is behaviorally independent in adults.

    Science.gov (United States)

    Ross, Elliott D; Gupta, Smita S; Adnan, Asif M; Holden, Thomas L; Havlicek, Joseph; Radhakrishnan, Sridhar

    2016-03-01

    Facial expressions are described traditionally as monolithic entities. However, humans have the capacity to produce facial blends, in which the upper and lower face simultaneously display different emotional expressions. This, in turn, has led to the Component Theory of facial expressions. Recent neuroanatomical studies in monkeys have demonstrated that there are separate cortical motor areas for controlling the upper and lower face that, presumably, also occur in humans. The lower face is represented on the posterior ventrolateral surface of the frontal lobes in the primary motor and premotor cortices and the upper face is represented on the medial surface of the posterior frontal lobes in the supplementary motor and anterior cingulate cortices. Our laboratory has been engaged in a series of studies exploring the perception and production of facial blends. Using high-speed videography, we began measuring the temporal aspects of facial expressions to develop a more complete understanding of the neurophysiology underlying facial expressions and facial blends. The goal of the research presented here was to determine if spontaneous facial expressions in adults are predominantly monolithic or exhibit independent motor control of the upper and lower face. We found that spontaneous facial expressions are very complex and that the motor control of the upper and lower face is overwhelmingly independent, thus robustly supporting the Component Theory of facial expressions. Seemingly monolithic expressions, be they full facial or facial blends, are most likely the result of a timing coincident rather than a synchronous coordination between the ventrolateral and medial cortical motor areas responsible for controlling the lower and upper face, respectively. In addition, we found evidence that the right and left face may also exhibit independent motor control, thus supporting the concept that spontaneous facial expressions are organized predominantly across the horizontal facial

  4. POTATO GRANULE-BOUND STARCH SYNTHASE PROMOTER-CONTROLLED GUS EXPRESSION - REGULATION OF EXPRESSION AFTER TRANSIENT AND STABLE TRANSFORMATION

    NARCIS (Netherlands)

    VANDERSTEEGE, G; NIEBOER, M; SWAVING, J; TEMPELAAR, MJ

    1992-01-01

    Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the beta-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient

  5. MK3 controls Polycomb target gene expression via negative feedback on ERK

    Directory of Open Access Journals (Sweden)

    Prickaerts Peggy

    2012-08-01

    Full Text Available Abstract Background Gene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1. The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs, all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3, a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription. Results Our current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the

  6. Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon

    DEFF Research Database (Denmark)

    Eberl, L; Christiansen, Gunna; Molin, S;

    1996-01-01

    The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflag......The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate......, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation....

  7. Genetic control of eosinophilia in mice: gene(s) expressed in bone marrow-derived cells control high responsiveness

    Energy Technology Data Exchange (ETDEWEB)

    Vadas, M.A.

    1982-02-01

    A heterogeneity in the capacity of strains of mice to mount eosinophilia is described. BALB/c and C3H are eosinophil high responder strains (EO-HR) and CBA and A/J are eosinophil low responder strains (EO-LR), judged by the response of blood eosinophils to Ascaris suum, and the response of blood, bone marrow, and spleen eosinophils to keyhole limpet hemocyanin given 2 days after 150 mg/kg cyclophosphamide. Some of the gene(s) for high responsiveness appear to be dominant because (EO-HR x EO-LR)F/sub 1/ mice were intermediate to high responders. This gene is expressed in bone marrow-derived cells because radiation chimeras of the type EO-HR..-->..F/sub 1/ were high responders and EO-LR..-->..F/sub 1/ were low responders. This description of a genetic control of eosinophilia in mice may be useful in understanding the role of this cell in parasite immunity and allergy.

  8. Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

    Science.gov (United States)

    Matsuoka, Jun-Ichi; Ishizuna, Fumiko; Kurumisawa, Keigo; Morohashi, Kengo; Ogawa, Tetsuhiro; Hidaka, Makoto; Saito, Katsuharu; Ezawa, Tatsuhiro; Aono, Toshihiro

    2017-07-25

    R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies

  9. Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls

    DEFF Research Database (Denmark)

    Fei, Gao; Luo, Yonglun; Li, Shengting

    2011-01-01

    Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural...... breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation......-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls...

  10. FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans.

    Science.gov (United States)

    Minor, Paul J; He, Ting-Fang; Sohn, Chang Ho; Asthagiri, Anand R; Sternberg, Paul W

    2013-09-01

    The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

  11. CovR-controlled global regulation of gene expression in Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Alexander Dmitriev

    Full Text Available CovR/S is a two-component signal transduction system (TCS that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI. Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.

  12. Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development

    Science.gov (United States)

    Anderson, Kelly M.; Anderson, Douglas M.; McAnally, John R.; Shelton, John M.; Bassel-Duby, Rhonda; Olson, Eric N.

    2017-01-01

    HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes1–4. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality1,5. Hand2 expression is tightly regulated by upstream enhancers6,7 that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications8. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome9, the Uph–Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment. PMID:27783597

  13. Dynamic control of the complement system by modulated expression of regulatory proteins.

    Science.gov (United States)

    Thurman, Joshua M; Renner, Brandon

    2011-01-01

    The complement system serves many biological functions, including the eradication of invasive pathogens and the removal of damaged cells and immune-complexes. Uncontrolled complement activation causes injury to host cells, however, so adequate regulation of the system is essential. Control of the complement system is maintained by a group of cell surface and circulating proteins referred to as complement regulatory proteins. The expression of the cell surface complement regulatory proteins varies from tissue to tissue. Furthermore, specific cell types can upregulate or downregulate the expression of these proteins in response to a variety of signals or insults. Altered regulation of the complement regulatory proteins can have important effects on local complement activation. In some circumstances this can be beneficial, such as in the setting of certain infections. In other circumstances, however, this can be a cause of complement-mediated injury of the tissue. A full understanding of the mechanisms by which the complement system is modulated at the local level can have important implications for how we diagnose and treat a wide range of inflammatory diseases.

  14. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    Science.gov (United States)

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  15. PPARgamma-Dependent Control of Renin Expression: Molecular Mechanisms and Pathophysiological Relevance

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    Vladimir T. Todorov

    2013-01-01

    Full Text Available During the last years accumulating evidence demonstrated that the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma regulates the expression of renin gene and thus the overall renin production. This review summarizes the current knowledge of the transcriptional control of the renin gene by PPARgamma received from variety of models ranging from cell culture to transgenic animals. The molecular mechanisms of the PPARgamma action on renin are particularly interesting because they are featured by two newly described characteristics: one of them is the recently identified PPARgamma target sequence Pal3 which is specific for the human renin gene and mediates exceptionally high sensitivity to transactivation; the other is the potentiating effect of PPARgamma on the cAMP signaling in the renin-producing cells. Furthermore, I discuss the need for generating of additional transgenic animal models which are more appropriate with regard to the role of the PPARgamma-dependent regulation of the renin gene expression in human diseases such as arterial hypertension and metabolic syndrome.

  16. PPARgamma-Dependent Control of Renin Expression: Molecular Mechanisms and Pathophysiological Relevance.

    Science.gov (United States)

    Todorov, Vladimir T

    2013-01-01

    During the last years accumulating evidence demonstrated that the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates the expression of renin gene and thus the overall renin production. This review summarizes the current knowledge of the transcriptional control of the renin gene by PPARgamma received from variety of models ranging from cell culture to transgenic animals. The molecular mechanisms of the PPARgamma action on renin are particularly interesting because they are featured by two newly described characteristics: one of them is the recently identified PPARgamma target sequence Pal3 which is specific for the human renin gene and mediates exceptionally high sensitivity to transactivation; the other is the potentiating effect of PPARgamma on the cAMP signaling in the renin-producing cells. Furthermore, I discuss the need for generating of additional transgenic animal models which are more appropriate with regard to the role of the PPARgamma-dependent regulation of the renin gene expression in human diseases such as arterial hypertension and metabolic syndrome.

  17. Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development.

    Science.gov (United States)

    Anderson, Kelly M; Anderson, Douglas M; McAnally, John R; Shelton, John M; Bassel-Duby, Rhonda; Olson, Eric N

    2016-11-17

    HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality. Hand2 expression is tightly regulated by upstream enhancers that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome, the Uph-Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.

  18. Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice.

    Science.gov (United States)

    DeMayo, F J; Finegold, M J; Hansen, T N; Stanley, L A; Smith, B; Bullock, D W

    1991-08-01

    The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

  19. Controlling gene expression in mice with tetracycline: application in pigment cell research.

    Science.gov (United States)

    Shin, M K

    2000-10-01

    Genetic manipulation techniques are widely used in mice to study the functions of genes. The most common strategy for assessing in vivo function involves making irreversible changes in the genome by homologous recombination. To complement this approach, a number of systems have been developed that allow specific and controlled expression of a gene. One of the more versatile and promising systems is based on the tetracycline (tet) responsive bacterial tetracycline repressor (TetR). In recent years, the tet system has proven to be a valuable method for understanding the function of genes involved in a number of physiological processes, including mouse models for human diseases such as cancer and neurological and pigment disorders. This review will highlight the power and elegance of the tet system by focusing on its utility in the study of two pigment cell-related biological problems, the pathogenesis of melanomas and melanocyte development in the embryo.

  20. Aire controls gene expression in the thymic epithelium with ordered stochasticity

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    Meredith, Matthew; Zemmour, David; Mathis, Diane; Benoist, Christophe

    2015-01-01

    Aire controls immunologic tolerance by inducing the ectopic thymic expression of many tissue-specific genes, acting broadly by removing stops on the transcriptional machinery. To better understand Aire’s specificity, we performed single-cell RNAseq and DNA methylation analysis in Aire-sufficient and -deficient medullary epithelial cells (mTECs). Each of Aire’s target genes was induced in only a minority of mTECs, independently of DNA methylation patterns, as small inter-chromosomal gene clusters activated in concert in a proportion of mTECs. These microclusters differed between individual mice, and thus suggest an organization of the DNA or of the epigenome that results from stochastic determinism, but is bookmarked and stable through mTEC divisions, ensuring more effective presentation of self-antigens, and favoring diversity of self-tolerance between individuals. PMID:26237550

  1. RNA- and protein-mediated control of Listeria monocytogenes virulence gene expression

    Science.gov (United States)

    Lebreton, Alice; Cossart, Pascale

    2017-01-01

    ABSTRACT The model opportunistic pathogen Listeria monocytogenes has been the object of extensive research, aiming at understanding its ability to colonize diverse environmental niches and animal hosts. Bacterial transcriptomes in various conditions reflect this efficient adaptability. We review here our current knowledge of the mechanisms allowing L. monocytogenes to respond to environmental changes and trigger pathogenicity, with a special focus on RNA-mediated control of gene expression. We highlight how these studies have brought novel concepts in prokaryotic gene regulation, such as the ‘excludon’ where the 5′-UTR of a messenger also acts as an antisense regulator of an operon transcribed in opposite orientation, or the notion that riboswitches can regulate non-coding RNAs to integrate complex metabolic stimuli into regulatory networks. Overall, the Listeria model exemplifies that fine RNA tuners act together with master regulatory proteins to orchestrate appropriate transcriptional programmes. PMID:27217337

  2. Epigenetics in Apicomplexa: control of gene expression during cell cycle progression, differentiation and antigenic variation.

    Science.gov (United States)

    Hakimi, Mohamed-Ali; Deitsch, Kirk W

    2007-08-01

    Apicomplexan parasites are important disease causing organisms that infect both animals and humans, causing extensive health and economic damage to human populations, particularly those in the developing world. The ability to perform genetic crosses, to engineer transgenic parasites lines, and the wealth of information made available through recent genome sequencing projects have made the laboratory study of these parasites important not only for understanding the diseases that they cause, but also for gaining insights into basic biological processes. The control of gene expression and cellular differentiation are particularly interesting in these organisms, as the apparent lack of large families of recognizable transcription factors typically found in other eukaryotic organisms suggests that they may be unusually reliant on epigenetic mechanisms. Here we review recent advances in the study of epigenetic gene regulation in the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.

  3. PTTG expression in different experimental and human prolactinomas in relation to dopaminergic control of lactotropes

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    Bronstein Marcello D

    2007-01-01

    Full Text Available Abstract Background Pituitary tumor transforming gene (pttg is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Nevertheless, its expression in prolactinomas and its relation with the pituitary dopamine receptor 2 (D2R are not well defined. We sought to determine the pituitary level of pttg in three different experimental models of prolactinomas with altered dopaminergic control of the pituitary: the dopaminergic D2R knockout female mouse, the estrogen-treated rat, and the senescent female rat. These three models shared the characteristics of increased pituitary weight, hyperprolactinemia, lactotrope hyperplasia and reduced or absent dopaminergic action at the pituitary level. We also studied samples from human macroprolactinomas, which were characterized as responsive or resistant to dopamine agonist therapy. Results When compared to female wild-type mice, pituitaries from female D2R knockout mice had decreased PTTG concentration, while no difference in pttg mRNA level was found. In senescent rats no difference in pituitary PTTG protein expression was found when compared to young rats. But, in young female rats treated with a synthetic estrogen (Diethylstylbestrol, 20 mg PTTG protein expression was enhanced (P = 0.029. Therefore, in the three experimental models of prolactinomas, pituitary size was increased and there was hyperprolactinemia, but PTTG levels followed different patterns. Patients with macroprolactinomas were divided in those in which dopaminergic therapy normalized or failed to normalize prolactin levels (responsive and resistant, respectively. When pituitary pttg mRNA level was analyzed in these macroprolactinomas, no differences were found. We next analyzed estrogen action at the pituitary by measuring pituitary estrogen receptor α levels. The D2R knockout female mice have low estrogen levels and in accordance, pituitary estrogen receptors were increased (P

  4. Unpredictable Variable Prenatal Stress Programs Expression of Genes Involved in Appetite Control and Energy Expenditure

    Science.gov (United States)

    Moyer, E. L.; Al-Shayeb, B.; Baer, L. A.; Ronca, A. E.

    2016-01-01

    Exposure to stress in the womb shapes neurobiological and physiological outcomes of offspring in later life, including body weight regulation and metabolic profiles. Our previous work utilizing a centrifugation-induced hyper-gravity demonstrated significantly increased (8-15%) body mass in male, but not female, rats exposed throughout gestation to chronic 2-g from conception to birth. We reported a similar outcome in adult offspring exposed throughout gestation to Unpredictable Variable Prenatal Stress (UVPS). Here we examine gene expression changes and the plasma of animals treated with our UVPS model to identify a potential role for prenatal stress in this hypergravity programming effect. Specifically we focused on appetite control and energy expenditure pathways in prenatally stressed adult (90-day-old) male Sprague-Dawley rats.

  5. Genetic and epigenetic control of gene expression by CRISPR–Cas systems

    Science.gov (United States)

    Lo, Albert; Qi, Lei

    2017-01-01

    The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363

  6. Gene Expression Profile of Endotoxin-stimulated Leukocytes of the Term New Born: Control of Cytokine Gene Expression by Interleukin-10

    Science.gov (United States)

    Davidson, Dennis; Zaytseva, Alla; Miskolci, Veronika; Castro-Alcaraz, Susana; Vancurova, Ivana; Patel, Hardik

    2013-01-01

    Introduction Increasing evidence now supports the association between the fetal inflammatory response syndrome (FIRS) with the pathogenesis of preterm labor, intraventricular hemorrhage and bronchopulmonary dysplasia. Polymorphonuclear leukocyte (PMNs) and mononuclear cell (MONOs) infiltration of the placenta is associated with these disorders. The aim of this study was to reveal cell-specific differences in gene expression and cytokine release in response to endotoxin that would elucidate inflammatory control mechanisms in the newly born. Methods PMNs and MONOs were separately isolated from the same cord blood sample. A genome-wide microarray screened for gene expression and related pathways at 4 h of LPS stimulation (n = 5). RT-qPCR and ELISA were performed for selected cytokines at 4 h and 18 h of LPS stimulation. Results Compared to PMNs, MONOs had a greater diversity and more robust gene expression that included pro-inflammatory (PI) cytokines, chemokines and growth factors at 4 h. Only MONOs had genes changing expression (all up regulated including interleukin-10) that were clustered in the JAK/STAT pathway. Pre-incubation with IL-10 antibody, for LPS-stimulated MONOs, led to up regulated PI and IL-10 gene expression and release of PI cytokines after 4 h. Discussion The present study suggests a dominant role of MONO gene expression in control of the fetal inflammatory response syndrome at 4 hrs of LPS stimulation. LPS-stimulated MONOs but not PMNs of the newborn have the ability to inhibit PI cytokine gene expression by latent IL-10 release. PMID:23326478

  7. Gene expression profile of endotoxin-stimulated leukocytes of the term new born: control of cytokine gene expression by interleukin-10.

    Directory of Open Access Journals (Sweden)

    Dennis Davidson

    Full Text Available INTRODUCTION: Increasing evidence now supports the association between the fetal inflammatory response syndrome (FIRS with the pathogenesis of preterm labor, intraventricular hemorrhage and bronchopulmonary dysplasia. Polymorphonuclear leukocyte (PMNs and mononuclear cell (MONOs infiltration of the placenta is associated with these disorders. The aim of this study was to reveal cell-specific differences in gene expression and cytokine release in response to endotoxin that would elucidate inflammatory control mechanisms in the newly born. METHODS: PMNs and MONOs were separately isolated from the same cord blood sample. A genome-wide microarray screened for gene expression and related pathways at 4 h of LPS stimulation (n = 5. RT-qPCR and ELISA were performed for selected cytokines at 4 h and 18 h of LPS stimulation. RESULTS: Compared to PMNs, MONOs had a greater diversity and more robust gene expression that included pro-inflammatory (PI cytokines, chemokines and growth factors at 4 h. Only MONOs had genes changing expression (all up regulated including interleukin-10 that were clustered in the JAK/STAT pathway. Pre-incubation with IL-10 antibody, for LPS-stimulated MONOs, led to up regulated PI and IL-10 gene expression and release of PI cytokines after 4 h. DISCUSSION: The present study suggests a dominant role of MONO gene expression in control of the fetal inflammatory response syndrome at 4 hrs of LPS stimulation. LPS-stimulated MONOs but not PMNs of the newborn have the ability to inhibit PI cytokine gene expression by latent IL-10 release.

  8. RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

    Science.gov (United States)

    Weber, Gregor G; Kortmann, Jens; Narberhaus, Franz; Klose, Karl E

    2014-09-30

    Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

  9. Quality control in microarray assessment of gene expression in human airway epithelium

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    Attiyeh Marc A

    2009-10-01

    Full Text Available Abstract Background Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223 of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC criteria established, included: (1 RNA quality, assessed by RNA Integrity Number (RIN ≥ 7.0; (2 cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets ≤ 3.0; and (3 the multi-chip normalization scaling factor ≤ 10.0. Results Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3% passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6% passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 ± 0.04 were significantly lower (p Conclusion Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.

  10. CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.

    Science.gov (United States)

    Li, Qi; Chen, Jun; Minton, Nigel P; Zhang, Ying; Wen, Zhiqiang; Liu, Jinle; Yang, Haifeng; Zeng, Zhe; Ren, Xiaodan; Yang, Junjie; Gu, Yang; Jiang, Weihong; Jiang, Yu; Yang, Sheng

    2016-07-01

    Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Temperature control of molecular circuit switch responsible for virulent phenotype expression in uropathogenic Escherichia coli

    Science.gov (United States)

    Samoilov, Michael

    2010-03-01

    The behavior and fate of biological organisms are to a large extent dictated by their environment, which can be often viewed as a collection of features and constraints governed by physics laws. Since biological systems comprise networks of molecular interactions, one such key physical property is temperature, whose variations directly affect the rates of biochemical reactions involved. For instance, temperature is known to control many gene regulatory circuits responsible for pathogenicity in bacteria. One such example is type 1 fimbriae (T1F) -- the foremost virulence factor in uropathogenic E. coli (UPEC), which accounts for 80-90% of all community-acquired urinary tract infections (UTIs). The expression of T1F is randomly `phase variable', i.e. individual cells switch between virulent/fimbriate and avirulent/afimbriate phenotypes, with rates regulated by temperature. Our computational investigation of this process, which is based on FimB/FimE recombinase-mediated inversion of fimS DNA element, offers new insights into its discrete-stochastic kinetics. In particular, it elucidates the logic of T1F control optimization to the host temperature and contributes further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.

  12. Probing the Limits to MicroRNA-Mediated Control of Gene Expression.

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    Araks Martirosyan

    2016-01-01

    Full Text Available According to the 'ceRNA hypothesis', microRNAs (miRNAs may act as mediators of an effective positive interaction between long coding or non-coding RNA molecules, carrying significant potential implications for a variety of biological processes. Here, inspired by recent work providing a quantitative description of small regulatory elements as information-conveying channels, we characterize the effectiveness of miRNA-mediated regulation in terms of the optimal information flow achievable between modulator (transcription factors and target nodes (long RNAs. Our findings show that, while a sufficiently large degree of target derepression is needed to activate miRNA-mediated transmission, (a in case of differential mechanisms of complex processing and/or transcriptional capabilities, regulation by a post-transcriptional miRNA-channel can outperform that achieved through direct transcriptional control; moreover, (b in the presence of large populations of weakly interacting miRNA molecules the extra noise coming from titration disappears, allowing the miRNA-channel to process information as effectively as the direct channel. These observations establish the limits of miRNA-mediated post-transcriptional cross-talk and suggest that, besides providing a degree of noise buffering, this type of control may be effectively employed in cells both as a failsafe mechanism and as a preferential fine tuner of gene expression, pointing to the specific situations in which each of these functionalities is maximized.

  13. Optimal control of gene expression for fast proteome adaptation to environmental change.

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    Pavlov, Michael Y; Ehrenberg, Måns

    2013-12-17

    Bacterial populations growing in a changing world must adjust their proteome composition in response to alterations in the environment. Rapid proteome responses to growth medium changes are expected to increase the average growth rate and fitness value of these populations. Little is known about the dynamics of proteome change, e.g., whether bacteria use optimal strategies of gene expression for rapid proteome adjustments and if there are lower bounds to the time of proteome adaptation in response to growth medium changes. To begin answering these types of questions, we modeled growing bacteria as stoichiometrically coupled networks of metabolic pathways. These are balanced during steady-state growth in a constant environment but are initially unbalanced after rapid medium shifts due to a shortage of enzymes required at higher concentrations in the new environment. We identified an optimal strategy for rapid proteome adjustment in the absence of protein degradation and found a lower bound to the time of proteome adaptation after medium shifts. This minimal time is determined by the ratio between the Kullback-Leibler distance from the pre- to the postshift proteome and the postshift steady-state growth rate. The dynamics of optimally controlled proteome adaptation has a simple analytical solution. We used detailed numerical modeling to demonstrate that realistic bacterial control systems can emulate this optimal strategy for rapid proteome adaptation. Our results may provide a conceptual link between the physiology and population genetics of growing bacteria.

  14. The physics of protein-DNA interaction networks in the control of gene expression

    Science.gov (United States)

    Saiz, Leonor

    2012-05-01

    Protein-DNA interaction networks play a central role in many fundamental cellular processes. In gene regulation, physical interactions and reactions among the molecular components together with the physical properties of DNA control how genes are turned on and off. A key player in all these processes is the inherent flexibility of DNA, which provides an avenue for long-range interactions between distal DNA elements through DNA looping. Such versatility enables multiple interactions and results in additional complexity that is remarkably difficult to address with traditional approaches. This topical review considers recent advances in statistical physics methods to study the assembly of protein-DNA complexes with loops, their effects in the control of gene expression, and their explicit application to the prototypical lac operon genetic system of the E. coli bacterium. In the last decade, it has been shown that the underlying physical properties of DNA looping can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including the balance between robustness and sensitivity of the induction process. These physical properties are largely dependent on the free energy of DNA looping, which accounts for DNA bending and twisting effects. These new physical methods have also been used in reverse to uncover the actual in vivo free energy of looping double-stranded DNA in living cells, which was not possible with existing experimental techniques. The results obtained for DNA looping by the lac repressor inside the E. coli bacterium showed a more malleable DNA than expected as a result of the interplay of the simultaneous presence of two distinct conformations of looped DNA.

  15. Pituitary control of branchial NCC, NKCC and Na(+), K (+)-ATPase α-subunit gene expression in Nile tilapia, Oreochromis niloticus.

    Science.gov (United States)

    Breves, Jason P; Seale, Andre P; Moorman, Benjamin P; Lerner, Darren T; Moriyama, Shunsuke; Hopkins, Kevin D; Grau, E Gordon

    2014-05-01

    This study investigated endocrine control of branchial ionoregulatory function in Nile tilapia (Oreochromis niloticus) by prolactin (Prl188 and Prl177), growth hormone (Gh) and cortisol. Branchial expression of Na(+)/Cl(-) cotransporter (ncc) and Na(+)/K(+)/2Cl(-) cotransporter (nkcc) genes were employed as specific markers for freshwater- and seawater-type ionocytes, respectively. We further investigated whether Prl, Gh and cortisol direct expression of two Na(+), K(+)-ATPase (nka)-α1 subunit genes, denoted nka-α1a and nka-α1b. Tilapia transferred to fresh water following hypophysectomy failed to adequately activate gill ncc expression; ncc expression was subsequently restored by Prl replacement. Prl188 and Prl177 stimulated ncc expression in cultured gill filaments in a concentration-related manner, suggesting that ncc is regulated by Prl in a gill-autonomous fashion. Tilapia transferred to brackish water (23 ‰) following hypophysectomy exhibited a reduced capacity to up-regulate nka-α1b expression. However, Gh and cortisol failed to affect nka-α1b expression in vivo. Similarly, we found no clear effects of Gh or cortisol on nkcc expression both in vivo and in vitro. When considered with patterns previously described in euryhaline Mozambique tilapia (O. mossambicus), the current study suggests that ncc is a conserved target of Prl in tilapiine cichlids. In addition, we revealed contrasting dependencies upon the pituitary to direct nka-α1b expression in hyperosmotic environments between Nile and Mozambique tilapia.

  16. Somatosensory Modulation of Salivary Gene Expression and Oral Feeding in Preterm Infants: Randomized Controlled Trial.

    Science.gov (United States)

    Barlow, Steven Michael; Maron, Jill Lamanna; Alterovitz, Gil; Song, Dongli; Wilson, Bernard Joseph; Jegatheesan, Priya; Govindaswami, Balaji; Lee, Jaehoon; Rosner, Austin Oder

    2017-06-14

    Despite numerous medical advances in the care of at-risk preterm neonates, oral feeding still represents one of the first and most advanced neurological challenges facing this delicate population. Objective, quantitative, and noninvasive assessment tools, as well as neurotherapeutic strategies, are greatly needed in order to improve feeding and developmental outcomes. Pulsed pneumatic orocutaneous stimulation has been shown to improve nonnutritive sucking (NNS) skills in preterm infants who exhibit delayed or disordered nipple feeding behaviors. Separately, the study of the salivary transcriptome in neonates has helped identify biomarkers directly linked to successful neonatal oral feeding behavior. The combination of noninvasive treatment strategies and transcriptomic analysis represents an integrative approach to oral feeding in which rapid technological advances and personalized transcriptomics can safely and noninvasively be brought to the bedside to inform medical care decisions and improve care and outcomes. The study aimed to conduct a multicenter randomized control trial (RCT) to combine molecular and behavioral methods in an experimental conceptualization approach to map the effects of PULSED somatosensory stimulation on salivary gene expression in the context of the acquisition of oral feeding habits in high-risk human neonates. The aims of this study represent the first attempt to combine noninvasive treatment strategies and transcriptomic assessments of high-risk extremely preterm infants (EPI) to (1) improve oral feeding behavior and skills, (2) further our understanding of the gene ontology of biologically diverse pathways related to oral feeding, (3) use gene expression data to personalize neonatal care and individualize treatment strategies and timing interventions, and (4) improve long-term developmental outcomes. A total of 180 extremely preterm infants from three neonatal intensive care units (NICUs) will be randomized to receive either PULSED or

  17. Evaluating methods for ranking differentially expressed genes applied to microArray quality control data

    Directory of Open Access Journals (Sweden)

    Shimizu Kentaro

    2011-06-01

    Full Text Available Abstract Background Statistical methods for ranking differentially expressed genes (DEGs from gene expression data should be evaluated with regard to high sensitivity, specificity, and reproducibility. In our previous studies, we evaluated eight gene ranking methods applied to only Affymetrix GeneChip data. A more general evaluation that also includes other microarray platforms, such as the Agilent or Illumina systems, is desirable for determining which methods are suitable for each platform and which method has better inter-platform reproducibility. Results We compared the eight gene ranking methods using the MicroArray Quality Control (MAQC datasets produced by five manufacturers: Affymetrix, Applied Biosystems, Agilent, GE Healthcare, and Illumina. The area under the curve (AUC was used as a measure for both sensitivity and specificity. Although the highest AUC values can vary with the definition of "true" DEGs, the best methods were, in most cases, either the weighted average difference (WAD, rank products (RP, or intensity-based moderated t statistic (ibmT. The percentages of overlapping genes (POGs across different test sites were mainly evaluated as a measure for both intra- and inter-platform reproducibility. The POG values for WAD were the highest overall, irrespective of the choice of microarray platform. The high intra- and inter-platform reproducibility of WAD was also observed at a higher biological function level. Conclusion These results for the five microarray platforms were consistent with our previous ones based on 36 real experimental datasets measured using the Affymetrix platform. Thus, recommendations made using the MAQC benchmark data might be universally applicable.

  18. Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene.

    Science.gov (United States)

    Guarneros, G; Montañez, C; Hernandez, T; Court, D

    1982-01-01

    The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. lambda mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hef mutations cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription of the int gene. There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection. Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis. PMID:6281759

  19. Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls.

    Directory of Open Access Journals (Sweden)

    Fei Gao

    Full Text Available Animal breeding via Somatic Cell Nuclear Transfer (SCNT has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF. Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver, using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.

  20. Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle

    OpenAIRE

    Mardaryev, Andrei N.; Ahmed, Mohammed I.; Vlahov, Nikola V.; Fessing, Michael Y.; Gill, Jason H.; Sharov, Andrey A.; Botchkareva, Natalia V.

    2010-01-01

    The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases duri...

  1. Relations among mothers' expressivity, children's effortful control, and their problem behaviors: a four-year longitudinal study.

    Science.gov (United States)

    Valiente, Carlos; Eisenberg, Nancy; Spinrad, Tracy L; Reiser, Mark; Cumberland, Amanda; Losoya, Sandra H; Liew, Jeffrey

    2006-08-01

    Longitudinal relations between mothers' expressivity, children's effortful control, and their problem behaviors were examined when children (N = 181) were 6.5-10 years old (T2) and again 2 (T3) and 4 (T4) years later. Mothers reported on their expression of positive and negative dominant emotion. Mothers and teachers reported on children's effortful control and externalizing and internalizing problem behaviors. In structural equation models, variables exhibited consistency over time. Further, the relation between mothers' expressivity (positive minus negative dominant emotion) at T2 and children's externalizing problems at T4 was mediated by T3 effortful control. The same process of mediation was significant for teacher- but not mother-reported internalizing problems. The results provide one explanation for how emotion-related socializing behaviors influence children's problem behaviors.

  2. Randomized Controlled Trial of Online Expressive Writing to Address Readjustment Difficulties Among U.S. Afghanistan and Iraq War Veterans.

    Science.gov (United States)

    Sayer, Nina A; Noorbaloochi, Siamak; Frazier, Patricia A; Pennebaker, James W; Orazem, Robert J; Schnurr, Paula P; Murdoch, Maureen; Carlson, Kathleen F; Gravely, Amy; Litz, Brett T

    2015-10-01

    We examined the efficacy of a brief, accessible, nonstigmatizing online intervention-writing expressively about transitioning to civilian life. U.S. Afghanistan and Iraq war veterans with self-reported reintegration difficulty (N = 1,292, 39.3% female, M = 36.87, SD = 9.78 years) were randomly assigned to expressive writing (n = 508), factual control writing (n = 507), or no writing (n = 277). Using intention to treat, generalized linear mixed models demonstrated that 6-months postintervention, veterans who wrote expressively experienced greater reductions in physical complaints, anger, and distress compared with veterans who wrote factually (ds = 0.13 to 0.20; ps reintegration difficulty compared with veterans who did not write at all (ds = 0.22 to 0.35; ps ≤ .001). Veterans who wrote expressively also experienced greater improvement in social support compared to those who did not write (d = 0.17). Relative to both control conditions, expressive writing did not lead to improved life satisfaction. Secondary analyses also found beneficial effects of expressive writing on clinically significant distress, PTSD screening, and employment status. Online expressive writing holds promise for improving health and functioning among veterans experiencing reintegration difficulty, albeit with small effect sizes.

  3. Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain

    NARCIS (Netherlands)

    D. Moechars (Dieder); M.C. Weston (Matthew); S. Leo (Sandra); Z. Callaerts-Vegh (Zsuzsanna); I. Goris (Ilse); R.F. Daneels; A. Buist (A.); M. Cik (M.); P.J. van der Spek (Peter); R.S. Kass (Robert); T. Meert (Theo); R. D'Hooge (Rudi); C. Rosenmund (Christian); R.M. Hampson (R. Mark)

    2006-01-01

    textabstractUptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus,

  4. Contributions of extracellular matrix signaling and tissue architecture to nuclear mechanisms and spatial organization of gene expression control.

    Science.gov (United States)

    Lelièvre, Sophie A

    2009-09-01

    Post-translational modification of histones, ATP-dependent chromatin remodeling, and DNA methylation are interconnected nuclear mechanisms that ultimately lead to the changes in chromatin structure necessary to carry out epigenetic gene expression control. Tissue differentiation is characterized by a specific gene expression profile in association with the acquisition of a defined tissue architecture and function. Elements critical for tissue differentiation, like extracellular stimuli, adhesion and cell shape properties, and transcription factors all contribute to the modulation of gene expression and thus, are likely to impinge on the nuclear mechanisms of epigenetic gene expression control. In this review, we analyze how these elements modify chromatin structure in a hierarchical manner by acting on the nuclear machinery. We discuss how mechanotransduction via the structural continuum of the cell and biochemical signaling to the cell nucleus integrate to provide a comprehensive control of gene expression. The role of nuclear organization in this control is highlighted, with a presentation of differentiation-induced nuclear structure and the concept of nuclear organization as a modulator of the response to incoming signals.

  5. Assessment of Control Tissue for Gene and Protein Expression Studies: A Comparison of Three Alternative Lung Sources

    Directory of Open Access Journals (Sweden)

    Margaret R. Passmore

    2012-01-01

    Full Text Available The use of an appropriate control group in human research is essential in investigating the level of a pathological disorder. This study aimed to compare three alternative sources of control lung tissue and to determine their suitability for gene and protein expression studies. Gene and protein expression levels of the vascular endothelial growth factor (VEGF and gelatinase families and their receptors were measured using real-time reverse transcription polymerase chain reaction (RT-PCR and immunohistochemistry. The gene expression levels of VEGFA, placental growth factor (PGF, and their receptors, fms-related tyrosine kinase 1 (FLT1, and kinase insert domain receptor (KDR as well as matrix metalloproteinase-2 (MMP-2 and the inhibitors, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1 and TIMP-2 were significantly higher in lung cancer resections. The gene expression level of MMP-9 was significantly lower in the corresponding samples. Altered protein expression was also detected, depending on the area assessed. The results of this study show that none of the three control groups studied are completely suitable for gene and protein studies associated with the VEGF and gelatinase families, highlighting the need for researchers to be selective in which controls they opt for.

  6. AMO EXPRESS: A Command and Control Experiment for Crew Autonomy Onboard the International Space Station

    Science.gov (United States)

    Stetson, Howard K.; Haddock, Angie T.; Frank, Jeremy; Cornelius, Randy; Wang, Lui; Garner, Larry

    2015-01-01

    NASA is investigating a range of future human spaceflight missions, including both Mars-distance and Near Earth Object (NEO) targets. Of significant importance for these missions is the balance between crew autonomy and vehicle automation. As distance from Earth results in increasing communication delays, future crews need both the capability and authority to independently make decisions. However, small crews cannot take on all functions performed by ground today, and so vehicles must be more automated to reduce the crew workload for such missions. NASA's Advanced Exploration Systems Program funded Autonomous Mission Operations (AMO) project conducted an autonomous command and control experiment on-board the International Space Station that demonstrated single action intelligent procedures for crew command and control. The target problem was to enable crew initialization of a facility class rack with power and thermal interfaces, and involving core and payload command and telemetry processing, without support from ground controllers. This autonomous operations capability is enabling in scenarios such as initialization of a medical facility to respond to a crew medical emergency, and representative of other spacecraft autonomy challenges. The experiment was conducted using the Expedite the Processing of Experiments for Space Station (EXPRESS) rack 7, which was located in the Port 2 location within the U.S Laboratory onboard the International Space Station (ISS). Activation and deactivation of this facility is time consuming and operationally intensive, requiring coordination of three flight control positions, 47 nominal steps, 57 commands, 276 telemetry checks, and coordination of multiple ISS systems (both core and payload). Utilization of Draper Laboratory's Timeliner software, deployed on-board the ISS within the Command and Control (C&C) computers and the Payload computers, allowed development of the automated procedures specific to ISS without having to certify

  7. Expression of bvg-repressed genes in Bordetella pertussis is controlled by RisA through a novel c-di-GMP signaling pathway

    Science.gov (United States)

    The BvgAS two component system of Bordetella pertussis controls virulence factor expression. In addition, BvgAS controls expression of the bvg-repressed genes through the action of the repressor, BvgR. The transcription factor RisA is inhibited by BvgR, and when BvgR is not expressed RisA induces th...

  8. Epigenetic control of Ccr7 expression in distinct lineages of lung dendritic cells.

    Science.gov (United States)

    Moran, Timothy P; Nakano, Hideki; Kondilis-Mangum, Hrisavgi D; Wade, Paul A; Cook, Donald N

    2014-11-15

    Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.

  9. Adiponectin expression in patients with inflammatory cardiomyopathy indicates favourable outcome and inflammation control.

    Science.gov (United States)

    Bobbert, Peter; Scheibenbogen, Carmen; Jenke, Alexander; Kania, Gabriele; Wilk, Sabrina; Krohn, Stefanie; Stehr, Jenny; Kuehl, Uwe; Rauch, Ursula; Eriksson, Urs; Schultheiss, Heinz Peter; Poller, Wolfgang; Skurk, Carsten

    2011-05-01

    Circulating adiponectin (APN) is an immunomodulatory, pro-angiogenic, and anti-apoptotic adipocytokine protecting against acute viral heart disease and preventing pathological remodelling after cardiac injury. The purpose of this study was to describe the regulation and effects of APN in patients with inflammatory cardiomyopathy (DCMi). Adiponectin expression and outcome were assessed in 173 patients with DCMi, 30 patients with non-inflammatory DCM, and 30 controls. Mechanistic background of these findings was addressed in murine experimental autoimmune myocarditis (EAM), a model of human DCMi, and further elucidated in vitro. Adiponectin plasma concentrations were significantly higher in DCMi compared with DCM or controls, i.e. 6.8 ± 3.9 µg/mL vs. 5.4 ± 3.6 vs. 4.76 ± 2.5 µg/mL (P< 0.05, respectively) and correlated significantly with cardiac mononuclear infiltrates (CD3+: r(2)= 0.025, P= 0.038; CD45R0+: r(2)= 0.058, P= 0.018). At follow-up, DCMi patients with high APN levels showed significantly increased left ventricular ejection fraction improvement, decreased left ventricular end-diastolic diameter, and reduced cardiac inflammatory infiltrates compared with patients with low APN levels. A multivariate linear regression analysis implicated APN as an independent prognostic factor for inhibition of cardiac inflammation. In accordance with these findings in human DCMi, EAM mice exhibited elevated plasma APN. Adiponectin gene transfer led to significant downregulation of key inflammatory mediators promoting disease. Mechanistically, APN acted as a negative regulator of T cells by reducing antigen specific expansion (P< 0.01) and suppressed TNFα-mediated NFκB activation (P< 0.01) as well as release of reactive oxygen species in cardiomyocytes. Our results implicate that APN acts as endogenously upregulated anti-inflammatory cytokine confining cardiac inflammation and progression in DCMi.

  10. The critical role of RNA processing and degradation in the control of gene expression.

    Science.gov (United States)

    Arraiano, Cecília M; Andrade, José M; Domingues, Susana; Guinote, Inês B; Malecki, Michal; Matos, Rute G; Moreira, Ricardo N; Pobre, Vânia; Reis, Filipa P; Saramago, Margarida; Silva, Inês J; Viegas, Sandra C

    2010-09-01

    The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

  11. TGF-β-induced IκB-ζ controls Foxp3 gene expression

    Energy Technology Data Exchange (ETDEWEB)

    MaruYama, Takashi, E-mail: ta-maru@umin.ac.jp [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578 (Japan); School of Medicine, Gifu University, Gifu 501-1194 (Japan)

    2015-08-21

    Inhibitor of kappa B (IκB)-ζ, a member of the nuclear IκB family of proteins, is induced by the transforming growth factor (TGF)-β signaling pathway and plays a pivotal role in maintaining the balance of T helper (Th) cell subsets. IκB-ζ deficiency results in reduced percentages of Th17 cells and increased percentages of Th1 cells. In this study, the effects of IκB-ζ deficiency on T-cell subsets were examined further. The data showed that IκB-ζ-deficient T cells had a high capacity for generation of regulatory T cells (Tregs) when T cells were cultured under TGF-β stimulation in the presence of cytokine-neutralizing antibodies. Mechanistically, IκB-ζ itself negatively regulated activation of the Foxp3 promoter in a nuclear factor of kappaB-dependent manner. Thus, this study showed that IκB-ζ controlled Treg differentiation. - Highlights: • IκB-ζ-deficient T cells exhibited increased generation of Foxp3{sup +} Tregs. • IκB-ζ played a key role in Foxp3 gene expression. • Retroviral overexpression of IκB-ζ was achieved in T cells.

  12. The Daily Lives of People With HIV Infection: A Qualitative Study of the Control Group in an Expressive Writing Intervention.

    Science.gov (United States)

    Metaweh, Maria; Ironson, Gail; Barroso, Julie

    2016-01-01

    Emotional disclosure is an expressive writing technique used in psychotherapy to process traumatic and stressful life experiences. While emotional disclosure interventions frequently use control groups, there are few qualitative analyses of these control groups. Our study's purpose was to analyze the control essays written by HIV-infected informants about their daily activities in an augmented written emotional disclosure intervention. Latent and manifest qualitative content analyses revealed prevalent contextual themes within the data. The emergent themes were socioeconomic status (SES), self-care, religiosity/spirituality, and social support. Emotional disclosure control subjects contributed substantial findings in terms of SES, self-care, resiliency, religiosity/spirituality, and social support and altruism.

  13. (Im)Perfect robustness and adaptation of metabolic networks subject to metabolic and gene-expression regulation: marrying control engineering with metabolic control analysis.

    Science.gov (United States)

    He, Fei; Fromion, Vincent; Westerhoff, Hans V

    2013-11-21

    Metabolic control analysis (MCA) and supply-demand theory have led to appreciable understanding of the systems properties of metabolic networks that are subject exclusively to metabolic regulation. Supply-demand theory has not yet considered gene-expression regulation explicitly whilst a variant of MCA, i.e. Hierarchical Control Analysis (HCA), has done so. Existing analyses based on control engineering approaches have not been very explicit about whether metabolic or gene-expression regulation would be involved, but designed different ways in which regulation could be organized, with the potential of causing adaptation to be perfect. This study integrates control engineering and classical MCA augmented with supply-demand theory and HCA. Because gene-expression regulation involves time integration, it is identified as a natural instantiation of the 'integral control' (or near integral control) known in control engineering. This study then focuses on robustness against and adaptation to perturbations of process activities in the network, which could result from environmental perturbations, mutations or slow noise. It is shown however that this type of 'integral control' should rarely be expected to lead to the 'perfect adaptation': although the gene-expression regulation increases the robustness of important metabolite concentrations, it rarely makes them infinitely robust. For perfect adaptation to occur, the protein degradation reactions should be zero order in the concentration of the protein, which may be rare biologically for cells growing steadily. A proposed new framework integrating the methodologies of control engineering and metabolic and hierarchical control analysis, improves the understanding of biological systems that are regulated both metabolically and by gene expression. In particular, the new approach enables one to address the issue whether the intracellular biochemical networks that have been and are being identified by genomics and systems

  14. A pipeline to determine RT-QPCR control genes for evolutionary studies: application to primate gene expression across multiple tissues.

    Directory of Open Access Journals (Sweden)

    Olivier Fedrigo

    Full Text Available Because many species-specific phenotypic differences are assumed to be caused by differential regulation of gene expression, many recent investigations have focused on measuring transcript abundance. Despite the availability of high-throughput platforms, quantitative real-time polymerase chain reaction (RT-QPCR is often the method of choice because of its low cost and wider dynamic range. However, the accuracy of this technique heavily relies on the use of multiple valid control genes for normalization. We created a pipeline for choosing genes potentially useful as RT-QPCR control genes for measuring expression between human and chimpanzee samples across multiple tissues, using published microarrays and a measure of tissue-specificity. We identified 13 genes from the pipeline and from commonly used control genes: ACTB, USP49, ARGHGEF2, GSK3A, TBP, SDHA, EIF2B2, GPDH, YWHAZ, HPTR1, RPL13A, HMBS, and EEF2. We then tested these candidate genes and validated their expression stability across species. We established the rank order of the most preferable set of genes for single and combined tissues. Our results suggest that for at least three tissues (cerebral cortex, liver, and skeletal muscle, EIF2B2, EEF2, HMBS, and SDHA are useful genes for normalizing human and chimpanzee expression using RT-QPCR. Interestingly, other commonly used control genes, including TBP, GAPDH, and, especially ACTB do not perform as well. This pipeline could be easily adapted to other species for which expression data exist, providing taxonomically appropriate control genes for comparisons of gene expression among species.

  15. RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation

    Science.gov (United States)

    Bouhlal, Hicham; Singh, Amrathlal Rabbind; Ossart, Christèle; Reignier, Aline; Hocini, Hakim; Fouquet, Gregory; Baghami, Mohammed Al; Eugenio, Mélanie Simoes; Nguyen-Khac, Eric; Regimbeau, Jean-Marc; Marcq, Ingrid

    2016-01-01

    Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression. PMID:26799423

  16. Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression.

    Science.gov (United States)

    Alami, R; Bender, M A; Feng, Y Q; Fiering, S N; Hug, B A; Ley, T J; Groudine, M; Bouhassira, E E

    2000-02-01

    The mouse beta-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the beta(min) and beta(maj) genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.

  17. Vitamin D analogs differentially control antimicrobial peptide/"alarmin" expression in psoriasis.

    Directory of Open Access Journals (Sweden)

    Mark Peric

    Full Text Available Antimicrobial peptides (AMPs are strongly expressed in lesional skin in psoriasis and play an important role as proinflammatory "alarmins" in this chronic skin disease. Vitamin D analogs like calcipotriol have antipsoriatic effects and might mediate this effect by changing AMP expression. In this study, keratinocytes in lesional psoriatic plaques showed decreased expression of the AMPs beta-defensin (HBD 2 and HBD3 after topical treatment with calcipotriol. At the same time, calcipotriol normalized the proinflammatory cytokine milieu and decreased interleukin (IL-17A, IL-17F and IL-8 transcript abundance in lesional psoriatic skin. In contrast, cathelicidin antimicrobial peptide expression was increased by calcipotriol while psoriasin expression remained unchanged. In cultured human epidermal keratinocytes the effect of different vitamin D analogs on the expression of AMPs was further analyzed. All vitamin D analogs tested blocked IL-17A induced HBD2 expression by increasing IkappaB-alpha protein and inhibition of NF-kappaB signaling. At the same time vitamin D analogs induced cathelicidin through activation of the vitamin D receptor and MEK/ERK signaling. These studies suggest that vitamin D analogs differentially alter AMP expression in lesional psoriatic skin and cultured keratinocytes. Balancing AMP "alarmin" expression might be a novel goal in treatment of chronic inflammatory skin diseases.

  18. Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhong-Min GUO; Kang XU; Ying YUE; Bing HUANG; Xin-Yan DENG; Nü-Qi ZHONG; Xun HONG; Xi-Gu CHEN; Dong XIAO

    2005-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTAtetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur.Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

  19. Methods of, and Reasons for, Emotional Expression and Control in Children with Internalizing, Externalizing, and Somatic Problems in Urban India

    Science.gov (United States)

    Raval, Vaishali V.; Martini, Tanya S.; Raval, Pratiksha H.

    2010-01-01

    Although cross-cultural research concerning children's emotions is growing, few studies have examined emotion dysregulation in culturally diverse populations. This study compared 6- to 8-year-old children's reported methods of expressing and controlling anger, sadness, and physical pain, and their justifications for doing so across four groups in…

  20. No sex diference in contextual control over the expression of latent inhibition and extinction in pavlovian fear conditioning in rats

    NARCIS (Netherlands)

    Maes, J.H.R.

    2002-01-01

    Three experiments with Wistar rats searched for a sex difference in contextual control over the expression of latent inhibition and extinction. Experiment 1 used a latent inhibition procedure; Experiments 2 and 3 employed an extinction preparation. All experiments used a shock as the unconditioned s

  1. Semi-active Attitude Control and Off-line Attitude Determination for the SEETI-Express Student Micro-satellite

    DEFF Research Database (Denmark)

    Alminde, Lars

    This paper concerns the development of the Attitude Determination and Control System (ADCS) for the SSETI-Express micro-satellite mission. The mission is an educational project involving 14 universities and the European Space Agency (ESA). The satellite has been designed and built, by students...

  2. Semi-active Attitude Control and Off-line Attitude Determination for the SSETI-Express Student Micro-satellite

    DEFF Research Database (Denmark)

    Alminde, Lars

    2005-01-01

    This paper concerns the development of the Attitude Determination and Control System (ADCS) for the SSETI-Express micro-satellite mission. The mission is an educational project involving 14 universities and the European Space Agency (ESA). The satellite has been designed and built, by students...

  3. Variation of ATM protein expression in response to irradiation of lymphocytes in lung cancer patients and controls.

    Science.gov (United States)

    Jianlin, Lou; Jiliang, He; Lifen, Jin; Wei, Zheng; Zhijian, Chen; Shijie, Chen; Shijie, Xu

    2006-07-05

    The aim of this research work was to study the cellular response to ionizing radiation (IR) and its relationship with the ATM protein expression levels in lung cancer patients. Heparinized blood samples were collected from 22 controls and 22 lung cancer patients. Each sample was divided into two parts: non-irradiated sample and irradiated sample, which was exposed to 3 Gy X-ray. The spontaneous and IR-induced genetic damage in both lung cancer patients and controls was measured with comet assay and micronucleus (MN) assay, and the ATM protein expression levels of non-irradiated samples in lung cancer patients and controls were detected by Western blotting. The results indicated that the baseline values of average mean tail moment (MTM) and micronucleus rate (MNR) in lung cancer patients were 0.86 and 11.41 per thousand, respectively, which was significantly higher than those (0.64 and 6.77 per thousand) of controls (P<0.05 for MTM, P<0.01 for MNR). The IR-induced average MTM and MNR in lung cancer patients were 1.23 and 77.64 per thousand, respectively, which was also significantly higher than those (0.71 and 66.05 per thousand) of controls (P<0.05 for MTM, P<0.01 for MNR). The results of Western blotting showed that the ATM protein expression levels in lung cancer patients and controls were 0.64 and 1.71, respectively, and there was significant (P<0.01) difference between lung cancer patients and controls. In present investigation, it was found that the genetic instability measured with comet assay and MN assay in lung cancer patients were significantly higher than those in controls, on the contrary, ATM protein expression level in lung cancer patients were significantly lower than that in controls. However, no good correlation was found either between ATM protein expression and IR-induced MTM or between ATM protein expression and IR-induced MNR in lung cancer patients.

  4. Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette

    Directory of Open Access Journals (Sweden)

    Zwick Friederike

    2012-10-01

    Full Text Available Abstract Background The XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli. Activation of transcription from the Pm promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements xylS (X, Pm (P and the 5'-untranslated region (U that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels. Results We combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene bla (resistance to penicillin. Combination of optimized elements stimulated bla expression up to 75-fold (X, P and U combined relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, celB (encoding phosphoglucomutase and the human growth factor gene gm-csf. Protein production from these genes is much more efficient than from bla in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for bla. We also integrated a single copy of the expression cassette with each gene into the E. coli chromosome and found that the expression level from this single copy was higher for bla than for the wild-type plasmid system, while it was lower for celB and gm-csf. Conclusion Our results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in E. coli. For one reporter gene (bla this allowed for more protein production from a single gene copy

  5. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

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    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  6. Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels.

    Science.gov (United States)

    Melly, Ludovic F; Marsano, Anna; Frobert, Aurelien; Boccardo, Stefano; Helmrich, Uta; Heberer, Michael; Eckstein, Friedrich S; Carrel, Thierry P; Giraud, Marie-Noëlle; Tevaearai, Hendrik T; Banfi, Andrea

    2012-10-01

    Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

  7. Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria

    Directory of Open Access Journals (Sweden)

    Harashima Hirofumi

    2012-06-01

    Full Text Available Abstract Background Cell proliferation is an important determinant of plant growth and development. In addition, modulation of cell-division rate is an important mechanism of plant plasticity and is key in adapting of plants to environmental conditions. One of the greatest challenges in understanding the cell cycle of flowering plants is the large families of CDKs and cyclins that have the potential to form many different complexes. However, it is largely unclear which complexes are active. In addition, there are many CDK- and cyclin-related proteins whose biological role is still unclear, i.e. whether they have indeed enzymatic activity. Thus, a biochemical characterization of these proteins is of key importance for the understanding of their function. Results Here we present a straightforward system to systematically express and purify active CDK-cyclin complexes from E. coli extracts. Our method relies on the concomitant production of a CDK activating kinase, which catalyzes the T-loop phosphorylation necessary for kinase activity. Taking the examples of the G1-phase cyclin CYCLIN D3;1 (CYCD3;1, the mitotic cyclin CYCLIN B1;2 (CYCB1;2 and the atypical meiotic cyclin SOLO DANCERS (SDS in conjunction with A-, B1- and B2-type CDKs, we show that different CDKs can interact with various cyclins in vitro but only a few specific complexes have high levels of kinase activity. Conclusions Our work shows that both the cyclin as well as the CDK partner contribute to substrate specificity in plants. These findings refine the interaction networks in cell-cycle control and pinpoint to particular complexes for modulating cell proliferation activity in breeding.

  8. WISP-1 positively regulates angiogenesis by controlling VEGF-A expression in human osteosarcoma.

    Science.gov (United States)

    Tsai, Hsiao-Chi; Tzeng, Huey-En; Huang, Chun-Yin; Huang, Yuan-Li; Tsai, Chun-Hao; Wang, Shih-Wei; Wang, Po-Chuan; Chang, An-Chen; Fong, Yi-Chin; Tang, Chih-Hsin

    2017-04-13

    In recent years, much research has focused on the role of angiogenesis in osteosarcoma, which occurs predominantly in adolescents and young adults. The vascular endothelial growth factor-A (VEGF-A) pathway is the key regulator of angiogenesis and in osteosarcoma. VEGF-A expression has been recognized as a prognostic marker in angiogenesis. Aberrant WNT1-inducible signaling pathway protein-1 (WISP-1) expression is associated with various cancers. However, the function of WISP-1 in osteosarcoma angiogenesis is poorly understood. We demonstrate a positive correlation between WISP-1 and VEGF-A expression in human osteosarcoma. Moreover, we show that WISP-1 promotes VEGF-A expression in human osteosarcoma cells, subsequently inducing human endothelial progenitor cell (EPC) migration and tube formation. The focal adhesion kinase (FAK), Jun amino-terminal kinase (JNK), and hypoxia-inducible factor (HIF)-1α signaling pathways were activated after WISP-1 stimulation, while FAK, JNK, and HIF-1α inhibitors or small interfering RNA (siRNA) abolished WISP-1-induced VEGF-A expression and angiogenesis. In vitro and in vivo studies revealed down-regulation of microRNA-381 (miR-381) in WISP-1-induced VEGF-A expression and angiogenesis. Our findings reveal that WISP-1 enhances VEGF-A expression and angiogenesis through the FAK/JNK/HIF-1α signaling pathways, as well as via down-regulation of miR-381 expression. WISP-1 may be a promising target in osteosarcoma angiogenesis.

  9. ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

    DEFF Research Database (Denmark)

    Marcos, Nuno T; Bennett, Eric P; Gomes, Joana

    2011-01-01

    found that ST6GalNAc-I is weakly expressed in normal gastric mucosa, but over-expressed in intestinal metaplasia, co-localized with sialyl-Tn. In gastric carcinomas ST6GalNAc-I was also associated with sialyl-Tn, but with heterogeneous staining and partial co-localization. Our results showed ST6Gal...

  10. Translational control of SCL-isoform expression in hematopoietic lineage choice

    NARCIS (Netherlands)

    Calkhoven, Cornelis F; Muller, Christine; Martin, Richard; Krosl, Goradz; Pietsch, Hubertus; Hoang, Trang; Leutz, Achim

    2003-01-01

    We investigated the translational regulation of SCL protein expression and its role in hematopoietic lineage choice. We show that the expression of different SCL protein isoforms is regulated by signal transduction pathways that modulate translation initiation factor (eIF) function. A conserved smal

  11. Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system

    NARCIS (Netherlands)

    Krzeslak, Joanna; Gerritse, Gijs; van Merkerk, Ronald; Cool, Robbert H.; Quax, Wim J.

    2008-01-01

    Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set

  12. Circulating adiponectin levels and expression of adiponectin receptors in relation to lung cancer: two case-control studies.

    Science.gov (United States)

    Petridou, Eleni T; Mitsiades, Nicholas; Gialamas, Spyros; Angelopoulos, Miltiadis; Skalkidou, Alkistis; Dessypris, Nick; Hsi, Alex; Lazaris, Nikolaos; Polyzos, Aristidis; Syrigos, Constantinos; Brennan, Aoife M; Tseleni-Balafouta, Sofia; Mantzoros, Christos S

    2007-01-01

    Decreased circulating levels of adiponectin, an adipocyte-secreted hormone and endogenous insulin sensitizer, have been associated with several obesity-related malignancies. Thiazolidinedione administration, which increases adiponectin levels, decreases risk for lung cancer. Whether circulating adiponectin levels are associated with lung cancer and/or whether adiponectin receptors are expressed in lung cancer remains unknown. We conducted a case-control study of 85 patients with incidental, histologically confirmed lung cancer and 170 healthy controls matched by gender and age. In a separate study, archival lung specimens from 134 cancerous and 8 noncancerous tissues were examined for relative expression of adiponectin receptors AdipoR1 and AdipoR2 using immunohistochemistry. Tobacco smoking, heavy alcohol intake and education were all associated with lung cancer risk, whereas serum adiponectin levels were not significantly different between cases and controls (multiple logistic regression, odds ratio per SD of adiponectin among controls: 1.13, 95% confidence interval: 0.64-2.02). Adiponectin levels were significantly lower (odds ratio: 0.25, 95% confidence interval: 0.10-0.78) among patients with advanced compared to those with limited disease stage. Expression of adiponectin receptors was apparent only in the cancerous lung tissue (64.2% AdipoR1 and 61.9% AdipoR2 in cancerous vs. 0% among noncancerous tissue). Specifically, AdipoR1 was expressed in all disease types, but no difference was noted with disease stage, whereas AdipoR2 was mainly expressed in the non-small cell carcinomas and more prominently in the advanced disease stage (80%). Circulating adiponectin levels are not different in cases of this malignancy - which seems to be unrelated to obesity and insulin resistance - compared to their healthy controls, though hormonal levels were significantly lower in advanced versus limited lung cancer. Both adiponectin receptors were expressed in cancerous lung

  13. Secretory expression of human protein in the Yeast Pichia pastoris by controlled fermentor culture.

    Science.gov (United States)

    Murasugi, Akira

    2010-06-01

    The heterologous protein expression system of Pichia pastoris is now widely used for expression of many human proteins, because the efficiently expressed proteins will be correctly folded in Pichia pastoris cells and also efficiently secreted from the cells. Recombinant human serum albumin (rHSA) is efficiently secreted from Pichia pastoris. Nowadays, the expression of rHSA exceeds 10g in 1 L fermentor culture broth, and the protein is completely purified. Recombinant HSA expressed in Pichia pastoris was approved as a medicine by the authorities in 2007, and launched in 2008 in Japan. One of the insulin precursors (IP) was also successfully expressed in Pichia pastoris, and secreted up to 3.6g in 1 L medium using a multi-copy transformant. The insulin precursor could be efficiently converted to insulin, the final product, in vitro. Human growth hormone was also expressed in Pichia pastoris, and secreted up to 49 mg in 1 L medium. These proteins are also important for clinical applications. Midkine and pleiotrophin may be two of the candidates for clinical applications. Secretion signals, the copy number of an expression cassette in transformants, and culture conditions for fermentation were examined for efficient expression of these proteins in Pichia pastoris. The best signal was selected, and other factors were optimized. The amounts of native midkine and native pleiotrophin expressed were approximately 0.36g and 0.26g in 1 L medium, respectively. Expression of bile-salt stimulated lipase (BSSL) had been extremely low in the beginning of a fermentor culture experiment. However, approximately 1 g rBSSL in 1 L medium was finally expressed in a fermentor by unlimited feeding of glycerol for cell growth and optimization of other factors. BSSL from human milk and rBSSL from Pichia cells are glycosylated. The structure differences between these glycans are obvious. When humanization of Pichia glycans is established by genetic engineering, the Pichia pastoris expression

  14. Distinct metabolic network states manifest in the gene expression profiles of pediatric inflammatory bowel disease patients and controls

    Science.gov (United States)

    Knecht, Carolin; Fretter, Christoph; Rosenstiel, Philip; Krawczak, Michael; Hütt, Marc-Thorsten

    2016-09-01

    Information on biological networks can greatly facilitate the function-orientated interpretation of high-throughput molecular data. Genome-wide metabolic network models of human cells, in particular, can be employed to contextualize gene expression profiles of patients with the goal of both, a better understanding of individual etiologies and an educated reclassification of (clinically defined) phenotypes. We analyzed publicly available expression profiles of intestinal tissues from treatment-naive pediatric inflammatory bowel disease (IBD) patients and age-matched control individuals, using a reaction-centric metabolic network derived from the Recon2 model. By way of defining a measure of ‘coherence’, we quantified how well individual patterns of expression changes matched the metabolic network. We observed a bimodal distribution of metabolic network coherence in both patients and controls, albeit at notably different mixture probabilities. Multidimensional scaling analysis revealed a bisectional pattern as well that overlapped widely with the metabolic network-based results. Expression differences driving the observed bimodality were related to cellular transport of thiamine and bile acid metabolism, thereby highlighting the crosstalk between metabolism and other vital pathways. We demonstrated how classical data mining and network analysis can jointly identify biologically meaningful patterns in gene expression data.

  15. Stochastic fluctuations and distributed control of gene expression impact cellular memory.

    Directory of Open Access Journals (Sweden)

    Guillaume Corre

    Full Text Available Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.

  16. Intranasal oxytocin increases facial expressivity, but not ratings of trustworthiness, in patients with schizophrenia and healthy controls.

    Science.gov (United States)

    Woolley, J D; Chuang, B; Fussell, C; Scherer, S; Biagianti, B; Fulford, D; Mathalon, D H; Vinogradov, S

    2017-05-01

    Blunted facial affect is a common negative symptom of schizophrenia. Additionally, assessing the trustworthiness of faces is a social cognitive ability that is impaired in schizophrenia. Currently available pharmacological agents are ineffective at improving either of these symptoms, despite their clinical significance. The hypothalamic neuropeptide oxytocin has multiple prosocial effects when administered intranasally to healthy individuals and shows promise in decreasing negative symptoms and enhancing social cognition in schizophrenia. Although two small studies have investigated oxytocin's effects on ratings of facial trustworthiness in schizophrenia, its effects on facial expressivity have not been investigated in any population. We investigated the effects of oxytocin on facial emotional expressivity while participants performed a facial trustworthiness rating task in 33 individuals with schizophrenia and 35 age-matched healthy controls using a double-blind, placebo-controlled, cross-over design. Participants rated the trustworthiness of presented faces interspersed with emotionally evocative photographs while being video-recorded. Participants' facial expressivity in these videos was quantified by blind raters using a well-validated manualized approach (i.e. the Facial Expression Coding System; FACES). While oxytocin administration did not affect ratings of facial trustworthiness, it significantly increased facial expressivity in individuals with schizophrenia (Z = -2.33, p = 0.02) and at trend level in healthy controls (Z = -1.87, p = 0.06). These results demonstrate that oxytocin administration can increase facial expressivity in response to emotional stimuli and suggest that oxytocin may have the potential to serve as a treatment for blunted facial affect in schizophrenia.

  17. NLRC5 controls basal MHC class I gene expression in an MHC enhanceosome-dependent manner.

    Science.gov (United States)

    Neerincx, Andreas; Rodriguez, Galaxia M; Steimle, Viktor; Kufer, Thomas A

    2012-05-15

    Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHC expression and revealed NLRC5 as an important regulator of basal MHC class I expression.

  18. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

    Science.gov (United States)

    Ikari, Jun; Smith, Lynette M; Nelson, Amy J; Iwasawa, Shunichiro; Gunji, Yoko; Farid, Maha; Wang, Xingqi; Basma, Hesham; Feghali-Bostwick, Carol; Liu, Xiangde; DeMeo, Dawn L; Rennard, Stephen I

    2015-04-01

    In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

  19. Expression Profile of Genes Potentially Associated with Adequate Glycemic Control in Patients with Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Sâmia Cruz Tfaile Corbi

    2017-01-01

    Full Text Available Despite increasing research in type 2 diabetes mellitus (T2D, there are few studies showing the impact of the poor glycemic control on biological processes occurring in T2D. In order to identify potential genes related to poorly/well-controlled patients with T2D, our strategy of investigation included a primary screen by microarray (Human Genome U133 in a small group of individuals followed by an independent validation in a greater group using RT-qPCR. Ninety patients were divided as follows: poorly controlled T2D (G1, well-controlled T2D (G2, and normoglycemic individuals (G3. After using affy package in R, differentially expressed genes (DEGs were prospected as candidate genes potentially relevant for the glycemic control in T2D patients. After validation by RT-qPCR, the obtained DEGs were as follows—G1 + G2 versus G3: HLA-DQA1, SOS1, and BRCA2; G2 versus G1: ENO2, VAMP2, CCND3, CEBPD, LGALS12, AGBL5, MAP2K5, and PPAP2B; G2 versus G3: HLA-DQB1, MCM4, and SEC13; and G1 versus G3: PPIC. This demonstrated a systemic exacerbation of the gene expression related to immune response in T2D patients. Moreover, genes related to lipid metabolisms and DNA replication/repair were influenced by the glycemic control. In conclusion, this study pointed out candidate genes potentially associated with adequate glycemic control in T2D patients, contributing to the knowledge of how the glycemic control could systemically influence gene expression.

  20. Control of IGFBP-2 expression by steroids and peptide hormones in vertebrates

    Directory of Open Access Journals (Sweden)

    Andreas eHoeflich

    2014-04-01

    Full Text Available IGFBP-2 (1 has been described as a brain tumor oncogene (2 and is widely expressed in cancers from different origins (3-8. IGFBP-2 alone cannot cause malignant transformation, yet progression of brain tumors to higher grade (9 and even has been provided as a protective element in earlier stages of multistage colon carcinogenesis (10. Therefore, it is crucial to understand the factors that determine expression patterns of IGFBP-2 under normal and malignant conditions. The present review provides a comprehensive update of known factors that have an impact on expression of IGFBP-2.

  1. MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo.

    Science.gov (United States)

    Cavalli, Giulio; Hayashi, Masahiro; Jin, Ying; Yorgov, Daniel; Santorico, Stephanie A; Holcomb, Cherie; Rastrou, Melinda; Erlich, Henry; Tengesdal, Isak W; Dagna, Lorenzo; Neff, C Preston; Palmer, Brent E; Spritz, Richard A; Dinarello, Charles A

    2016-02-02

    Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an "adjuvant" during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity.

  2. The Polycomb-group protein MEDEA regulates seed development by controlling expression of the MADS-box gene PHERES1.

    Science.gov (United States)

    Köhler, Claudia; Hennig, Lars; Spillane, Charles; Pien, Stephane; Gruissem, Wilhelm; Grossniklaus, Ueli

    2003-06-15

    The Polycomb-group (PcG) proteins MEDEA, FERTILIZATION INDEPENDENT ENDOSPERM, and FERTILIZATION INDEPENDENT SEED2 regulate seed development in Arabidopsis by controlling embryo and endosperm proliferation. All three of these FIS-class proteins are likely subunits of a multiprotein PcG complex, which epigenetically regulates downstream target genes that were previously unknown. Here we show that the MADS-box gene PHERES1 (PHE1) is commonly deregulated in the fis-class mutants. PHE1 belongs to the evolutionarily ancient type I class of MADS-box proteins that have not yet been assigned any function in plants. Both MEDEA and FIE directly associate with the promoter region of PHE1, suggesting that PHE1 expression is epigenetically regulated by PcG proteins. PHE1 is expressed transiently after fertilization in both the embryo and the endosperm; however, it remains up-regulated in the fis mutants, consistent with the proposed function of the FIS genes as transcriptional repressors. Reduced expression levels of PHE1 in medea mutant seeds can suppress medea seed abortion, indicating a key role of PHE1 repression in seed development. PHE1 expression in a hypomethylated medea mutant background resembles the wild-type expression pattern and is associated with rescue of the medea seed-abortion phenotype. In summary, our results demonstrate that seed abortion in the medea mutant is largely mediated by deregulated expression of the type I MADS-box gene PHE1.

  3. Establishment of a new quality control and vaccine safety test for influenza vaccines and adjuvants using gene expression profiling.

    Science.gov (United States)

    Momose, Haruka; Mizukami, Takuo; Kuramitsu, Madoka; Takizawa, Kazuya; Masumi, Atsuko; Araki, Kumiko; Furuhata, Keiko; Yamaguchi, Kazunari; Hamaguchi, Isao

    2015-01-01

    We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.

  4. Brain-derived neurotrophic factor expression is higher in brain tissue from patients with refractory epilepsy than in normal controls

    Institute of Scientific and Technical Information of China (English)

    Yudan Lv; Jiqing Qiu; Zan Wang; Li Cui; Hongmei Meng; Weihong Lin

    2011-01-01

    The role of the brain-derived neurotrophic factor in epilepsy remains controversial. The present study utilized light and electron microscopy to investigate pathological and ultrastructural changes in brain tissue obtained from the seizure foci of 24 patients with temporal epilepsy. We found that epileptic tissue showed neuronal degeneration, glial cell proliferation, nuclear vacuolization, and neural cell tropism. Immunoelectron microscopy and immunohistochemistry showed that brain-derived neurotrophic factor was expressed at significantly higher levels in patients with refractory temporal epilepsy compared with normal controls, demonstrating that the pathological changes within seizure foci in patients with refractory epilepsy are associated with brain-derived neurotrophic factor expression alterations.

  5. Transcriptional programs that control expression of the autoimmune regulator gene Aire.

    Science.gov (United States)

    Herzig, Yonatan; Nevo, Shir; Bornstein, Chamutal; Brezis, Miriam R; Ben-Hur, Sharon; Shkedy, Aya; Eisenberg-Bord, Michal; Levi, Ben; Delacher, Michael; Goldfarb, Yael; David, Eyal; Weinberger, Leehee; Viukov, Sergey; Ben-Dor, Shifra; Giraud, Matthieu; Hanna, Jacob H; Breiling, Achim; Lyko, Frank; Amit, Ido; Feuerer, Markus; Abramson, Jakub

    2017-02-01

    Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.

  6. A wound-induced Wnt expression program controls planarian regeneration polarity.

    Science.gov (United States)

    Petersen, Christian P; Reddien, Peter W

    2009-10-06

    Regeneration requires specification of the identity of new tissues to be made. Whether this process relies only on intrinsic regulative properties of regenerating tissues or whether wound signaling provides input into tissue repatterning is not known. The head-versus-tail regeneration polarity decision in planarians, which requires Wnt signaling, provides a paradigm to study the process of tissue identity specification during regeneration. The Smed-wntP-1 gene is required for regeneration polarity and is expressed at the posterior pole of intact animals. Surprisingly, wntP-1 was expressed at both anterior- and posterior-facing wounds rapidly after wounding. wntP-1 expression was induced by all types of wounds examined, regardless of whether wounding prompted tail regeneration. Regeneration polarity was found to require new expression of wntP-1. Inhibition of the wntP-2 gene enhanced the polarity phenotype due to wntP-1 inhibition, with new expression of wntP-2 in regeneration occurring subsequent to expression of wntP-1 and localized only to posterior-facing wounds. New expression of wntP-2 required wound-induced wntP-1. Finally, wntP-1 and wntP-2 expression changes occurred even in the absence of neoblast stem cells, which are required for regeneration, suggesting that the role of these genes in polarity is independent of and instructive for tail formation. These data indicate that wound-induced input is involved in resetting the normal polarized features of the body axis during regeneration.

  7. Fat content of expressed breast milk: a case for quality control.

    OpenAIRE

    Spencer, S A; Hull, D

    1981-01-01

    Expressed breast milk used to feed preterm infants is precious and so, despite heterogeneity of composition, all available milk is used. A study of 274 samples of expressed breast milk supplied by preterm mothers and National Childbirth Trust donors showed pronounced variation in fat content as measured by the "creamatocrit" method. This was not due to differences between term and preterm mothers or between transitional and mature milk. The composition was affected by diurnal variation and me...

  8. Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells

    Science.gov (United States)

    De Cecco, Loris; Capaia, Matteo; Zupo, Simona; Cutrona, Giovanna; Matis, Serena; Brizzolara, Antonella; Orengo, Anna Maria; Croce, Michela; Marchesi, Edoardo; Ferrarini, Manlio; Canevari, Silvana; Ferrini, Silvano

    2015-01-01

    Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process. PMID:26305332

  9. Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Loris De Cecco

    Full Text Available Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes, whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

  10. A nucleotide metabolite controls stress-responsive gene expression and plant development

    KAUST Repository

    Chen, Hao

    2011-10-19

    Abiotic stress, such as drought and high salinity, activates a network of signaling cascades that lead to the expression of many stress-responsive genes in plants. The Arabidopsis FIERY1 (FRY1) protein is a negative regulator of stress and abscisic acid (ABA) signaling and exhibits both an inositol polyphosphatase and a 3?,5?-bisphosphate nucleotidase activity in vitro. The FRY1 nucleotidase degrades the sulfation byproduct 3?-phosphoadenosine-5?-phosphate (PAP), yet its in vivo functions and particularly its roles in stress gene regulation remain unclear. Here we developed a LC-MS/MS method to quantitatively measure PAP levels in plants and investigated the roles of this nucleotidase activity in stress response and plant development. It was found that PAP level was tightly controlled in plants and did not accumulate to any significant level either under normal conditions or under NaCl, LiCl, cold, or ABA treatments. In contrast, high levels of PAP were detected in multiple mutant alleles of FRY1 but not in mutants of other FRY1 family members, indicating that FRY1 is the major enzyme that hydrolyzes PAP in vivo. By genetically reducing PAP levels in fry1 mutants either through overexpression of a yeast PAP nucleotidase or by generating a triple mutant of fry1 apk1 apk2 that is defective in the biosynthesis of the PAP precursor 3?-phosphoadenosine-5?-phosphosulfate (PAPS), we demonstrated that the developmental defects and superinduction of stress-responsive genes in fry1 mutants correlate with PAP accumulation in planta. We also found that the hypersensitive stress gene regulation in fry1 requires ABH1 but not ABI1, two other negative regulators in ABA signaling pathways. Unlike in yeast, however, FRY1 overexpression in Arabidopsis could not enhance salt tolerance. Taken together, our results demonstrate that PAP is critical for stress gene regulation and plant development, yet the FRY1 nucleotidase that catabolizes PAP may not be an in vivo salt toxicity target

  11. A nucleotide metabolite controls stress-responsive gene expression and plant development.

    Directory of Open Access Journals (Sweden)

    Hao Chen

    Full Text Available Abiotic stress, such as drought and high salinity, activates a network of signaling cascades that lead to the expression of many stress-responsive genes in plants. The Arabidopsis FIERY1 (FRY1 protein is a negative regulator of stress and abscisic acid (ABA signaling and exhibits both an inositol polyphosphatase and a 3',5'-bisphosphate nucleotidase activity in vitro. The FRY1 nucleotidase degrades the sulfation byproduct 3'-phosphoadenosine-5'-phosphate (PAP, yet its in vivo functions and particularly its roles in stress gene regulation remain unclear. Here we developed a LC-MS/MS method to quantitatively measure PAP levels in plants and investigated the roles of this nucleotidase activity in stress response and plant development. It was found that PAP level was tightly controlled in plants and did not accumulate to any significant level either under normal conditions or under NaCl, LiCl, cold, or ABA treatments. In contrast, high levels of PAP were detected in multiple mutant alleles of FRY1 but not in mutants of other FRY1 family members, indicating that FRY1 is the major enzyme that hydrolyzes PAP in vivo. By genetically reducing PAP levels in fry1 mutants either through overexpression of a yeast PAP nucleotidase or by generating a triple mutant of fry1 apk1 apk2 that is defective in the biosynthesis of the PAP precursor 3'-phosphoadenosine-5'-phosphosulfate (PAPS, we demonstrated that the developmental defects and superinduction of stress-responsive genes in fry1 mutants correlate with PAP accumulation in planta. We also found that the hypersensitive stress gene regulation in fry1 requires ABH1 but not ABI1, two other negative regulators in ABA signaling pathways. Unlike in yeast, however, FRY1 overexpression in Arabidopsis could not enhance salt tolerance. Taken together, our results demonstrate that PAP is critical for stress gene regulation and plant development, yet the FRY1 nucleotidase that catabolizes PAP may not be an in vivo salt

  12. Dynamic expression of imprinted genes associates with maternally controlled nutrient allocation during maize endosperm development.

    Science.gov (United States)

    Xin, Mingming; Yang, Ruolin; Li, Guosheng; Chen, Hao; Laurie, John; Ma, Chuang; Wang, Dongfang; Yao, Yingyin; Larkins, Brian A; Sun, Qixin; Yadegari, Ramin; Wang, Xiangfeng; Ni, Zhongfu

    2013-09-01

    In angiosperms, the endosperm provides nutrients for embryogenesis and seed germination and is the primary tissue where gene imprinting occurs. To identify the imprintome of early developing maize (Zea mays) endosperm, we performed high-throughput transcriptome sequencing of whole kernels at 0, 3, and 5 d after pollination (DAP) and endosperms at 7, 10, and 15 DAP, using B73 by Mo17 reciprocal crosses. We observed gradually increased expression of paternal transcripts in 3- and 5-DAP kernels. In 7-DAP endosperm, the majority of the genes tested reached a 2:1 maternal versus paternal ratio, suggesting that paternal genes are nearly fully activated by 7 DAP. A total of 116, 234, and 63 genes exhibiting parent-specific expression were identified at 7, 10, and 15 DAP, respectively. The largest proportion of paternally expressed genes was at 7 DAP, mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP and were primarily attributed to sharply increased expression levels compared with the other stages. Gene ontology enrichment analysis of the imprinted genes suggested that 10-DAP endosperm-specific MEGs are involved in nutrient uptake and allocation and the auxin signaling pathway, coincident with the onset of starch and storage protein accumulation.

  13. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zeilstra, Jurrit; Joosten, Sander P.J. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Wensveen, Felix M. [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Dessing, Mark C.; Schuetze, Denise M. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Eldering, Eric [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Spaargaren, Marcel [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Pals, Steven T., E-mail: s.t.pals@amc.uva.nl [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)

    2011-03-04

    Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causes constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which

  14. MEK activity controls IL-8 expression in tamoxifen-resistant MCF-7 breast cancer cells.

    Science.gov (United States)

    Kim, Sangmin; Jeon, Myeongjin; Lee, Jeong Eon; Nam, Seok Jin

    2016-04-01

    Although tamoxifen reduces disease progression, tamoxifen resistance occurs during the course of estrogen receptor-positive [ER+] breast cancer treatment. In the present study, we investigated the possibility that interleukin-8 (IL-8) is a prognostic marker for tamoxifen resistance and aimed to clarify the regulation of IL-8 expression in tamoxifen-resistant cells. Clinically, IL-8 expression is positively correlated with survival in luminal A type breast cancer patients, but not in luminal B type breast cancer patients. In addition, the levels of IL-8 mRNA and protein expression were significantly increased in tamoxifen-resistant (TamR) cells compared to tamoxifen-sensitive (TamS) cells. To determine the regulatory mechanism of IL-8 expression in TamR cells, we analyzed the activities of signaling molecules. Our results showed that the phosphorylation levels of MEK and Akt were markedly increased in TamR cells, but there was no change in the phosphorylation level of p38 MAPK. On the contrary, we observed that elevated IL-8 mRNA expression was suppressed by a specific MEK1/2 inhibitor, UO126, but not by the specific PI-3K inhibitor LY294002, in TamR cells, whereas, we found that overexpression of constitutively active-MEK (CA-MEK) significantly increased the levels of IL-8 mRNA expression in TamS cells. Finally, we investigated the effect of the specific CXCR1/2 inhibitor SB225002 on anchorage-independent growth of TamR cells, and found that the growth was completely suppressed by SB225002. Taken together, our results demonstrate that IL-8 expression is regulated through a MEK/ERK-dependent pathway in TamR cells, suggesting that IL-8 and its receptors may be promising therapeutic targets for overcoming tamoxifen resistance.

  15. Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P.

    Science.gov (United States)

    Bruce, W; Folkerts, O; Garnaat, C; Crasta, O; Roth, B; Bowen, B

    2000-01-01

    To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol. The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr. The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.

  16. Altered Cytokine Gene Expression in Peripheral Blood Monocytes across the Menstrual Cycle in Primary Dysmenorrhea: A Case-Control Study

    Science.gov (United States)

    Ma, Hongyue; Hong, Min; Duan, Jinao; Liu, Pei; Fan, Xinsheng; Shang, Erxin; Su, Shulan; Guo, Jianming; Qian, Dawei; Tang, Yuping

    2013-01-01

    Primary dysmenorrhea is one of the most common gynecological complaints in young women, but potential peripheral immunologic features underlying this condition remain undefined. In this paper, we compared 84 common cytokine gene expression profiles of peripheral blood mononuclear cells (PBMCs) from six primary dysmenorrheic young women and three unaffected controls on the seventh day before (secretory phase), and the first (menstrual phase) and the fifth (regenerative phase) days of menstruation, using a real-time PCR array assay combined with pattern recognition and gene function annotation methods. Comparisons between dysmenorrhea and normal control groups identified 11 (nine increased and two decreased), 14 (five increased and nine decreased), and 15 (seven increased and eight decreased) genes with ≥2-fold difference in expression (Pmenstrual phase, genes encoding pro-inflammatory cytokines (IL1B, TNF, IL6, and IL8) were up-regulated, and genes encoding TGF-β superfamily members (BMP4, BMP6, GDF5, GDF11, LEFTY2, NODAL, and MSTN) were down-regulated. Functional annotation revealed an excessive inflammatory response and insufficient TGF-β superfamily member signals with anti-inflammatory consequences, which may directly contribute to menstrual pain. In the secretory and regenerative phases, increased expression of pro-inflammatory cytokines and decreased expression of growth factors were also observed. These factors may be involved in the regulation of decidualization, endometrium breakdown and repair, and indirectly exacerbate primary dysmenorrhea. This first study of cytokine gene expression profiles in PBMCs from young primary dysmenorrheic women demonstrates a shift in the balance between expression patterns of pro-inflammatory cytokines and TGF-β superfamily members across the whole menstrual cycle, underlying the peripheral immunologic features of primary dysmenorrhea. PMID:23390521

  17. Evaluating Light-Induced Promoters for the Control of Heterologous Gene Expression in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Albers, Stevan C; Peebles, Christie A M

    2017-01-01

    Cyanobacteria are enticing microbial factories, but little is understood how their gene control elements respond to the periodic availability to light. This research tested the capability of PpsbAII to control gene expression during light/dark conditions when moved to a neutral location within the Synechocystis sp. PCC 6803 genome. When the eYFP reporter gene was run by PpsbAII in the promoter's native genomic location, mutants exposed to 12-hour light conditions experienced a 15.8× increase in transcript abundance over that observed from the same construct exposed to 12-hour dark conditions. When this same construct was moved to the hypothetical coding region slr0168 in the genome, transcripts generated during 12 hour light conditions accumulated to 1.67X of the levels of transcripts generated by the same construct during 12 hour dark conditions. Three additional promoter constructs, PpsbAIII , PgroEL2 , and PsigD were also tested for differential expression in light and dark conditions within the neutral region slr0168. While low amounts of transcript accumulation were observed from PgroEL2 and PsigD , the PpsbAIII construct accumulated 5.79× more transcripts when compared to transcript abundance during dark conditions, which highlights the potential of this promoter to control gene expression during diel-cycle light conditions. Additionally, nucleotide mutations were made to regions within PpsbAII . Mutations to the cis-acting hexo-nucleotide region increased expression 3.71× over that of the native promoter, while the addition of the "HLR" nucleotide region to the PpsbAII::ΔHex construct increased expression 2.76× over that of the native promoter. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:45-53, 2017.

  18. ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

    DEFF Research Database (Denmark)

    Marcos, Nuno T; Bennett, Eric Paul; Gomes, Joana;

    2011-01-01

    Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc......-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl......-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we...

  19. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    Science.gov (United States)

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  20. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Anssi Rantasalo

    Full Text Available This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1 the transcription-activation domain of the sTF, 2 the binding-site modules in the output promoter, and 3 the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications.

  1. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

    Science.gov (United States)

    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  2. Small RNA-dependent expression of secondary metabolism is controlled by Krebs cycle function in Pseudomonas fluorescens.

    Science.gov (United States)

    Takeuchi, Kasumi; Kiefer, Patrick; Reimmann, Cornelia; Keel, Christoph; Dubuis, Christophe; Rolli, Joëlle; Vorholt, Julia A; Haas, Dieter

    2009-12-11

    Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.

  3. Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle.

    Science.gov (United States)

    Mardaryev, Andrei N; Ahmed, Mohammed I; Vlahov, Nikola V; Fessing, Michael Y; Gill, Jason H; Sharov, Andrey A; Botchkareva, Natalia V

    2010-10-01

    The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR-31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.

  4. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

    Science.gov (United States)

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-09-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

  5. An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b.

    Science.gov (United States)

    Mühmer, Mario; Ditthardt, Denise; Jäkel, Jörg; Wischmann, Viktoria; Moll, Roland; Schmidt, Ansgar

    2014-01-01

    The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.

  6. Environment control to improve recombinant protein yields in plants based on Agrobacterium-mediated transient gene expression

    Directory of Open Access Journals (Sweden)

    Naomichi eFujiuchi

    2016-03-01

    Full Text Available Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: 1 recombinant protein content per unit biomass; and 2 recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on those parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and post-inoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Pre-inoculation environmental factors associated with planting density, light quality and nutrient supply affect plant characteristics such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, post-inoculation environmental factors such as temperature, light intensity and humidity have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the post-inoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain

  7. Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance.

    Science.gov (United States)

    Stump, Madeliene; Guo, Deng-Fu; Lu, Ko-Ting; Mukohda, Masashi; Liu, Xuebo; Rahmouni, Kamal; Sigmund, Curt D

    2016-07-01

    Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions.

  8. Semi-active Attitude Control and Off-line Attitude Determination for the SSETI-Express Student Micro-satellite

    OpenAIRE

    Alminde, Lars

    2005-01-01

    This paper concerns the development of the Attitude Determination and Control System (ADCS) for the SSETI-Express micro-satellite mission. The mission is an educational project involving 14 universities and the European Space Agency (ESA). The satellite has been designed and built, by students, over a period of only 18 months. This paper emphasises on the trade-offs required to build an operational ADCS system within such a rapidly developing project.

  9. Changing expression of vertebrate immunity genes in an anthropogenic environment: a controlled experiment.

    Science.gov (United States)

    Hablützel, Pascal I; Brown, Martha; Friberg, Ida M; Jackson, Joseph A

    2016-09-01

    The effect of anthropogenic environments on the function of the vertebrate immune system is a problem of general importance. For example, it relates to the increasing rates of immunologically-based disease in modern human populations and to the desirability of identifying optimal immune function in domesticated animals. Despite this importance, our present understanding is compromised by a deficit of experimental studies that make adequately matched comparisons between wild and captive vertebrates. We transferred post-larval fishes (three-spined sticklebacks), collected in the wild, to an anthropogenic (captive) environment. We then monitored, over 11 months, how the systemic expression of immunity genes changed in comparison to cohort-matched wild individuals in the originator population (total n = 299). We found that a range of innate (lyz, defbl2, il1r-like, tbk1) and adaptive (cd8a, igmh) immunity genes were up-regulated in captivity, accompanied by an increase in expression of the antioxidant enzyme, gpx4a. For some genes previously known to show seasonality in the wild, this appeared to be reduced in captive fishes. Captive fishes tended to express immunity genes, including igzh, foxp3b, lyz, defbl2, and il1r-like, more variably. Furthermore, although gene co-expression patterns (analyzed through gene-by-gene correlations and mutual information theory based networks) shared common structure in wild and captive fishes, there was also significant divergence. For one gene in particular, defbl2, high expression was associated with adverse health outcomes in captive fishes. Taken together, these results demonstrate widespread regulatory changes in the immune system in captive populations, and that the expression of immunity genes is more constrained in the wild. An increase in constitutive systemic immune activity, such as we observed here, may alter the risk of immunopathology and contribute to variance in health in vertebrate populations exposed to

  10. Nipbl and mediator cooperatively regulate gene expression to control limb development.

    Directory of Open Access Journals (Sweden)

    Akihiko Muto

    2014-09-01

    Full Text Available Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS, the most common "cohesinopathy". It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb, knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.

  11. Gene expression profiling in persons with multiple chemical sensitivity before and after a controlled n-butanol exposure session

    DEFF Research Database (Denmark)

    Dantoft, Thomas Meinertz; Skovbjerg, Sine; Andersson, Linus

    2017-01-01

    To investigate the pathophysiological pathways leading to symptoms elicitation in multiple chemical sensitivity (MCS) by comparing gene expression in MCS participants and healthy controls before and after a chemical exposure optimised to cause symptoms among MCS participants.The first hypothesis...... min after being exposed to and 4 hours after the exposure. Participants suffering from MCS and healthy controls were recruited through advertisement at public places and in a local newspaper. 36 participants who considered themselves sensitive were prescreened for eligibility. 18 sensitive persons...

  12. Control of gene expression and mitochondrial biogenesis in the muscular adaption to endurance exercise

    DEFF Research Database (Denmark)

    Joseph, A. M.; Pilegaard, H.; Leick, L.

    2006-01-01

    Every time a bout of exercise is performed, a change in gene expression occurs within the contracting muscle. Over the course of many repeated bouts of exercise (i.e. training), the cumulative effects of these alterations lead to a change in muscle phenotype. One of the most prominent of these ad......Every time a bout of exercise is performed, a change in gene expression occurs within the contracting muscle. Over the course of many repeated bouts of exercise (i.e. training), the cumulative effects of these alterations lead to a change in muscle phenotype. One of the most prominent...

  13. Hanazonobashi facilities control system. Centralized SCADA for metropolitan express-way; Shutokosokudoro koden Kanagawa kanribudono osame. Hanazonokyo shisetsu kanri system

    Energy Technology Data Exchange (ETDEWEB)

    Higashino, K.; Imai, K.; Kitaura, M.; Kayama, C.; Tsujita, H. [Nisshin Electric Co. Ltd., Kyoto (Japan)

    1996-03-29

    This paper introduces the centralized SCADA system in the express-way line of whole Kanagawa region, which can control various facilities, such as power receiving, distribution and switch facilities, pump houses, transformer towers, and lighting facilities for bridges. The system was designed so as to monitor the whole of the Wangan line to be extended in the future. It consists of a central facility control station (CS) and local control stations (LS). The CS can control the whole facilities and the LS have control functions of start/stop for individual facilities at the maintenance and inspection of the CS. The SCADA system is surpassing in the operability of a huge amount of information, information exchange with other control systems and disaster preventing systems, and the extendability and maintainability. It is a distributed computer control system and also a multi-window type highly functional man-machine system. Multiple projection type large displays were employed to use information in common among operators. To support the facility maintenance works effectively, a data base has been made for collective control of information including drawings, facilities registers, and manual books. 9 figs., 1 tab.

  14. Auto and cross regulatory elements control Onecut expression in the ascidian nervous system.

    Science.gov (United States)

    Pezzotti, Maria Rosa; Locascio, Annamaria; Racioppi, Claudia; Fucci, Laura; Branno, Margherita

    2014-06-15

    The expression pattern of Onecut genes in the central and peripheral nervous systems is highly conserved in invertebrates and vertebrates but the regulatory networks in which they are involved are still largely unknown. The presence of three gene copies in vertebrates has revealed the functional roles of the Onecut genes in liver, pancreas and some populations of motor neurons. Urochordates have only one Onecut gene and are the closest living relatives of vertebrates and thus represent a good model system to understand its regulatory network and involvement in nervous system formation. In order to define the Onecut genetic cascade, we extensively characterized the Onecut upstream cis-regulatory DNA in the ascidian Ciona intestinalis. Electroporation experiments using a 2.5kb genomic fragment and of a series of deletion constructs identified a small region of 262bp able to reproduce most of the Onecut expression profile during embryonic development. Further analyses, both bioinformatic and in vivo using transient transgenes, permitted the identification of transcription factors responsible for Onecut endogenous expression. We provide evidence that Neurogenin is a direct activator of Onecut and that an autoregulatory loop is responsible for the maintenance of its expression. Furthermore, for the first time we propose the existence of a direct connection among Neurogenin, Onecut and Rx transcription factors in photoreceptor cell formation.

  15. Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A Randomized Controlled Trial

    Science.gov (United States)

    2008-03-01

    expression and fatty acid synthesis. Research in normal cells has demonstrated that dietary supplementation with polyunsaturated fatty acids ( PUFA ...particularly omega -3 fatty acids , inhibits SREBP-1 activation, resulting in a decreased transcription of FAS. 15. SUBJECT TERMS Prostate Cancer...Lipid Medtabolism, Clinical Trial; Omega -3 Fatty Acids 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME

  16. HDAC5 controls MEF2C-driven sclerostin expression in osteocytes.

    Science.gov (United States)

    Wein, Marc N; Spatz, Jordan; Nishimori, Shigeki; Doench, John; Root, David; Babij, Philip; Nagano, Kenichi; Baron, Roland; Brooks, Daniel; Bouxsein, Mary; Pajevic, Paola Divieti; Kronenberg, Henry M

    2015-03-01

    Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

  17. Grafting Acoustic Instruments and Signal Processing: Creative Control and Augmented Expressivity

    DEFF Research Database (Denmark)

    Overholt, Daniel; Freed, Adrian

    the musician’s movements. For example, gestural tilting of the instrument is tracked via an embedded Inertial Measurement Unit (IMU), which can be assigned to alter parameters such as the wet/dry mix of a simple octave-doubler or other more advanced audio effect, further augmenting the expressivity...

  18. SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer

    NARCIS (Netherlands)

    Papadakis, Andreas I; Sun, Chong; Knijnenburg, Theo A; Xue, Yibo; Grernrum, Wipawadee; Hölzel, Michael; Nijkamp, Wouter; Wessels, Lodewyk F A; Beijersbergen, Roderick L; Bernards, Rene; Huang, Sidong

    2015-01-01

    Recurrent inactivating mutations in components of SWI/SNF chromatin-remodeling complexes have been identified across cancer types, supporting their roles as tumor suppressors in modulating oncogenic signaling pathways. We report here that SMARCE1 loss induces EGFR expression and confers resistance t

  19. Control of transferrin expression by β-amyloid through the CP2 transcription factor.

    Science.gov (United States)

    Jang, Sang-Min; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Kang, Eun-Jin; Kim, Chul Geun; Kim, Hyun-Jung; Choi, Kyung-Hee

    2010-10-01

    Accumulation of β-amyloid protein (Aβ) is one of the most important pathological features of Alzheimer's disease. Although Aβ induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aβ-induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aβ (Aβ(1-42) and Aβ(25-35) ). An unbiased motif search of the transferrin promoter region showed that CP2 binds to the transferrin promoter, an iron-regulating protein, and regulates transferrin transcription. Ectopic expression of CP2 led to increased transferrin expression at both the mRNA and protein levels, whereas knockdown of CP2 down-regulated transferrin mRNA and protein expression. Moreover, CP2 trans-activated transcription of a transferrin reporter gene. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay showed that CP2 binds to the transferrin promoter region. Furthermore, the binding affinity of CP2 to the transferrin promoter was regulated by Aβ, as Aβ (Aβ(1-42) and Aβ(25-35) ) markedly increased the binding affinity of CP2 for the transferrin promoter. Taken together, these results suggest that CP2 contributes to the pathogenesis of Alzheimer's disease by inducing transferrin expression via up-regulating its transcription.

  20. Methotrexate increases skeletal muscle GLUT4 expression and improves metabolic control in experimental diabetes

    Science.gov (United States)

    Long-term administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) mimics the effects of endurance exercise by activating AMP kinase and by increasing skeletal muscle expression of GLUT4 glucose transporter. AICAR is an intermediate in the purine de novo synthesis, and its tissue conc...

  1. Nuclear Compartmentalization Contributes to Stage-Specific Gene Expression Control in Trypanosoma cruzi

    Science.gov (United States)

    Pastro, Lucía; Smircich, Pablo; Di Paolo, Andrés; Becco, Lorena; Duhagon, María A.; Sotelo-Silveira, José; Garat, Beatriz

    2017-01-01

    In the protozoan parasite Trypanosoma cruzi, as in other trypanosomatids, transcription of protein coding genes occurs in a constitutive fashion, producing large polycistronic transcription units. These units are composed of non-functionally related genes which are pervasively processed to yield each mRNA. Therefore, post-transcriptional processes are crucial to regulate gene expression. Considering that nuclear compartmentalization could contribute to gene expression regulation, we comparatively studied the nuclear, cytoplasmic and whole cell transcriptomes of the non-infective epimastigote stage of T. cruzi, using RNA-Seq. We found that the cytoplasmic transcriptome tightly correlates with the whole cell transcriptome and both equally correlate with the proteome. Nonetheless, 1,200 transcripts showed differential abundance between the nuclear and cytoplasmic fractions. For the genes with transcript content augmented in the nucleus, significant structural and compositional differences were found. The analysis of the reported epimastigote translatome and proteome, revealed scarce ribosome footprints and encoded proteins for them. Ontology analyses unveiled that many of these genes are distinctive of other parasite life-cycle stages. Finally, the relocalization of transcript abundance in the metacyclic trypomastigote infective stage was confirmed for specific genes. While gene expression is strongly dependent on transcript steady-state level, we here highlight the importance of the distribution of transcripts abundance between compartments in T. cruzi. Particularly, we show that nuclear compartmentation is playing an active role in the developmental stage determination preventing off-stage expression. PMID:28243589

  2. Control of gene expression and mitochondrial biogenesis in the muscular adaption to endurance exercise

    DEFF Research Database (Denmark)

    Joseph, A. M.; Pilegaard, H.; Leick, L.;

    2006-01-01

    Every time a bout of exercise is performed, a change in gene expression occurs within the contracting muscle. Over the course of many repeated bouts of exercise (i.e. training), the cumulative effects of these alterations lead to a change in muscle phenotype. One of the most prominent of these ad...

  3. Expression of mutant huntingtin in leptin receptor-expressing neurons does not control the metabolic and psychiatric phenotype of the BACHD mouse.

    Directory of Open Access Journals (Sweden)

    Sofia Hult Lundh

    Full Text Available Metabolic and psychiatric disturbances occur early on in the clinical manifestation of Huntington's disease (HD, a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT gene. Hypothalamus has emerged as an important site of pathology and alterations in this area and its neuroendocrine circuits may play a role in causing early non-motor symptoms and signs in HD. Leptin is a hormone that controls energy homeostasis by signaling through leptin receptors in the hypothalamus. Disturbed leptin action is implicated in both obesity and depression and altered circulating levels of leptin have been reported in both clinical HD and rodent models of the disease. Pathological leptin signaling may therefore be involved in causing the metabolic and psychiatric disturbances of HD. Here we tested the hypothesis that expression of mutant HTT in leptin receptor carrying neurons plays a role in the development of the non-motor phenotype in the BACHD mouse model. Our results show that inactivation of mutant HTT in leptin receptor-expressing neurons in the BACHD mouse using cross-breeding based on a cre-loxP system did not have an effect on the metabolic phenotype or anxiety-like behavior. The data suggest that mutant HTT disrupts critical hypothalamic pathways by other mechanisms than interfering with intracellular leptin signaling.

  4. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Directory of Open Access Journals (Sweden)

    Yue Yu

    Full Text Available BACKGROUND: Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. METHODS: Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. RESULTS: Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. CONCLUSIONS: Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  5. Estrogen controls vitamin D3-mediated resistance to experimental autoimmune encephalomyelitis by controlling vitamin D3 metabolism and receptor expression.

    Science.gov (United States)

    Nashold, Faye E; Spach, Karen M; Spanier, Justin A; Hayes, Colleen E

    2009-09-15

    Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease with a rapidly increasing female gender bias. MS prevalence decreases with increasing sunlight exposure, supporting our hypothesis that the sunlight-dependent hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is a natural inhibitor of autoimmune T cell responses in MS. We found that vitamin D(3) inhibited experimental autoimmune encephalomyelitis (EAE) in intact female mice, but not in ovariectomized females or males. To learn whether 17beta-estradiol (E(2)) is essential for vitamin D(3)-mediated protection, ovariectomized female mice were given E(2) or placebo and evaluated for vitamin D(3)-mediated EAE resistance. Diestrus-level E(2) implants alone provided no benefit, but they restored vitamin D(3)-mediated EAE resistance in the ovariectomized females. Synergy between E(2) and vitamin D(3) occurred through vitamin D(3)-mediated enhancement of E(2) synthesis, as well as E(2)-mediated enhancement of vitamin D receptor expression in the inflamed CNS. In males, E(2) implants did not enable vitamin D(3) to inhibit EAE. The finding that vitamin D(3)-mediated protection in EAE is female-specific and E(2)-dependent suggests that declining vitamin D(3) supplies due to sun avoidance might be contributing to the rapidly increasing female gender bias in MS. Moreover, declining E(2) synthesis and vitamin D(3)-mediated protection with increasing age might be contributing to MS disease progression in older women.

  6. The Nutrient-Dependent O-GlcNAc Modification Controls the Expression of Liver Fatty Acid Synthase.

    Science.gov (United States)

    Baldini, Steffi F; Wavelet, Cindy; Hainault, Isabelle; Guinez, Céline; Lefebvre, Tony

    2016-08-14

    Liver Fatty Acid Synthase (FAS) is pivotal for de novo lipogenesis. Loss of control of this metabolic pathway contributes to the development of liver pathologies ranging from steatosis to nonalcoholic steatohepatitis (NASH) which can lead to cirrhosis and, less frequently, to hepatocellular carcinoma. Therefore, deciphering the molecular mechanisms governing the expression and function of key enzymes such as FAS is crucial. Herein, we link the availability of this lipogenic enzyme to the nutrient-dependent post-translational modification O-GlcNAc that is thought to be deregulated in metabolic diseases (diabetes, obesity, and metabolic syndrome). We demonstrate that expression and activity of liver FAS correlate with O-GlcNAcylation contents in ob/ob mice and in mice fed with a high-carbohydrate diet both in a transcription-dependent and -independent manner. More importantly, inhibiting the removal of O-GlcNAc residues in mice intraperitoneally injected with the selective and potent O-GlcNAcase (OGA) inhibitor Thiamet-G increases FAS expression. FAS and O-GlcNAc transferase (OGT) physically interact, and FAS is O-GlcNAc modified. Treatment of a liver cell line with drugs or nutrients that elevate the O-GlcNAcylation interferes with FAS expression. Inhibition of OGA increases the interaction between FAS and the deubiquitinase Ubiquitin-specific protease-2a (USP2A) in vivo and ex vivo, providing mechanistic insights into the control of FAS expression through O-GlcNAcylation. Together, these results reveal a new type of regulation of FAS, linked to O-GlcNAcylation status, and advance our knowledge on deregulation of lipogenesis in diverse forms of liver diseases.

  7. Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002.

    Science.gov (United States)

    Zess, Erin K; Begemann, Matthew B; Pfleger, Brian F

    2016-02-01

    Predictive control of gene expression is an essential tool for developing synthetic biological systems. The current toolbox for controlling gene expression in cyanobacteria is a barrier to more in-depth genetic analysis and manipulation. Towards relieving this bottleneck, this work describes the use of synthetic biology to construct an anhydrotetracycline-based induction system and adapt a trans-acting small RNA (sRNA) system for use in the cyanobacterium Synechococcus sp. strain PCC 7002. An anhydrotetracycline-inducible promoter was developed to maximize intrinsic strength and dynamic range. The resulting construct, PEZtet , exhibited tight repression and a maximum 32-fold induction upon addition of anhydrotetracycline. Additionally, a sRNA system based on the Escherichia coli IS10 RNA-IN/OUT regulator was adapted for use in Synechococcus sp. strain PCC 7002. This system exhibited 70% attenuation of target gene expression, providing a demonstration of the use of sRNAs for differential gene expression in cyanobacteria. These systems were combined to produce an inducible sRNA system, which demonstrated 59% attenuation of target gene expression. Lastly, the role of Hfq, a critical component of sRNA systems in E. coli, was investigated. Genetic studies showed that the Hfq homolog in Synechococcus sp. strain PCC 7002 did not impact repression by the engineered sRNA system. In summary, this work describes new synthetic biology tools that can be applied to physiological studies, metabolic engineering, or sRNA platforms in Synechococcus sp. strain PCC 7002. © 2015 Wiley Periodicals, Inc.

  8. Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection.

    Science.gov (United States)

    Liu, Shuyan; Chen, Lingming; Zeng, Ying; Si, Lulu; Guo, Xiaolan; Zhou, Junmei; Fang, Danyun; Zeng, Gucheng; Jiang, Lifang

    2016-09-01

    Dengue virus (DENV) remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B (GrzB), may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a*, miR-30e, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer (NK) cells and CD8(+) T cells. Further investigation indicated that NK cells, but not CD8(+) T cells, were the major sources of GrzB, and miR-378, but not miR-27a* or miR-30e, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

  9. Multiple sclerosis: microRNA expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls.

    Directory of Open Access Journals (Sweden)

    Andreas Keller

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory demyelinating disease of the central nervous system, which is heterogenous with respect to clinical manifestations and response to therapy. Identification of biomarkers appears desirable for an improved diagnosis of MS as well as for monitoring of disease activity and treatment response. MicroRNAs (miRNAs are short non-coding RNAs, which have been shown to have the potential to serve as biomarkers for different human diseases, most notably cancer. Here, we analyzed the expression profiles of 866 human miRNAs. In detail, we investigated the miRNA expression in blood cells of 20 patients with relapsing-remitting MS (RRMS and 19 healthy controls using a human miRNA microarray and the Geniom Real Time Analyzer (GRTA platform. We identified 165 miRNAs that were significantly up- or downregulated in patients with RRMS as compared to healthy controls. The best single miRNA marker, hsa-miR-145, allowed discriminating MS from controls with a specificity of 89.5%, a sensitivity of 90.0%, and an accuracy of 89.7%. A set of 48 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 95%, a sensitivity of 97.6%, and an accuracy of 96.3%. While 43 of the 165 miRNAs deregulated in patients with MS have previously been related to other human diseases, the remaining 122 miRNAs are so far exclusively associated with MS. The implications of our study are twofold. The miRNA expression profiles in blood cells may serve as a biomarker for MS, and deregulation of miRNA expression may play a role in the pathogenesis of MS.

  10. New regulatory circuit controlling spatial and temporal gene expression in the sea urchin embryo oral ectoderm GRN.

    Science.gov (United States)

    Li, Enhu; Materna, Stefan C; Davidson, Eric H

    2013-10-01

    The sea urchin oral ectoderm gene regulatory network (GRN) model has increased in complexity as additional genes are added to it, revealing its multiple spatial regulatory state domains. The formation of the oral ectoderm begins with an oral-aboral redox gradient, which is interpreted by the cis-regulatory system of the nodal gene to cause its expression on the oral side of the embryo. Nodal signaling drives cohorts of regulatory genes within the oral ectoderm and its derived subdomains. Activation of these genes occurs sequentially, spanning the entire blastula stage. During this process the stomodeal subdomain emerges inside of the oral ectoderm, and bilateral subdomains defining the lateral portions of the future ciliary band emerge adjacent to the central oral ectoderm. Here we examine two regulatory genes encoding repressors, sip1 and ets4, which selectively prevent transcription of oral ectoderm genes until their expression is cleared from the oral ectoderm as an indirect consequence of Nodal signaling. We show that the timing of transcriptional de-repression of sip1 and ets4 targets which occurs upon their clearance explains the dynamics of oral ectoderm gene expression. In addition two other repressors, the direct Nodal target not, and the feed forward Nodal target goosecoid, repress expression of regulatory genes in the central animal oral ectoderm thereby confining their expression to the lateral domains of the animal ectoderm. These results have permitted construction of an enhanced animal ectoderm GRN model highlighting the repressive interactions providing precise temporal and spatial control of regulatory gene expression. © 2013 Elsevier Inc. All rights reserved.

  11. Preprocessing and Quality Control Strategies for Illumina DASL Assay-Based Brain Gene Expression Studies with Semi-Degraded Samples.

    Science.gov (United States)

    Chow, Maggie L; Winn, Mary E; Li, Hai-Ri; April, Craig; Wynshaw-Boris, Anthony; Fan, Jian-Bing; Fu, Xiang-Dong; Courchesne, Eric; Schork, Nicholas J

    2012-01-01

    Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples.

  12. Wilson protein expression, copper excretion and sweat production in sweat glands of Wilson disease patients and controls

    Directory of Open Access Journals (Sweden)

    Weiss Karl

    2008-07-01

    Full Text Available Abstract Background In Wilson disease, copper is not sufficiently excreted into bile due to the absence or malfunction of the Wilson protein copper ATPase in the excretory pathway of hepatocytes. Copper is found in sweat. It is unknown if the Wilson protein plays a role in copper excretion into sweat. It is the aim of this study to investigate Wilson protein expression in sweat glands and analysing its effects on copper excretion into sweat in controls and patients with Wilson disease. Methods Immunofluorescent analysis of the Wilson protein in skin samples from normal rat, LEC rat and human skin biopsies were performed. Pilocarpin-induced sweat gland stimulation by iontophoretic transfer adapted from the methods used for cystic fibrosis sweat test was used for sweat induction. Sweat volume, sweat copper concentration, serum ceruloplasmin and serum copper were analysed in 28 Wilson patients and 21 controls. Results The Wilson protein is expressed in human and rat sweat gland epithelia. Copper concentration in sweat is not significantly different between controls and Wilson patients. Wilson patients produce significantly smaller volumes of sweat compared to controls. Sweat production is partially reversible in Wilson patients under medical treatment for Wilson disease or after liver transplantation Conclusion Wilson patients show a reduced sweat production with unaltered sweat copper concentration. The Wilson protein might play an important role in physiological sweat production.

  13. EVEN-SKIPPED HOMEOBOX 1 controls human ES cell differentiation by directly repressing GOOSECOID expression

    DEFF Research Database (Denmark)

    Kalisz, Mark; Winzi, Maria Karin; Bisgaard, Hanne Cathrine;

    2012-01-01

    TGFß signaling patterns the primitive streak, yet little is known about transcriptional effectors that mediate the cell fate choices during streak-like development in mammalian embryos and in embryonic stem (ES) cells. Here we demonstrate that cross-antagonistic actions of EVEN-SKIPPED HOMEOBOX 1...... (EVX1) and GOOSECOID (GSC) regulate cell fate decisions in streak-like progenitors derived from human ES cells exposed to BMP4 and/or activin. We found that EVX1 repressed GSC expression and promoted formation of posterior streak-like progeny in response to BMP4, and conversely that GSC repressed EVX1...... expression and was required for development of anterior streak-like progeny in response to activin. Chromatin immunoprecipitation assays showed that EVX1 bound to the GSC 5'-flanking region in BMP4 treated human ES cells, and band shift assays identified two EVX1 binding sites in the GSC 5'-region...

  14. RAE-1 is expressed in the adult subventricular zone and controls cell proliferation of neurospheres

    DEFF Research Database (Denmark)

    Popa, Natalia; Cédile, Oriane; Pollet-Villard, Xavier

    2011-01-01

    playing either immune or nonimmune function. Among the latter, MHC functions in the central nervous system has started to receive recent interest. Here, our first goal was to investigate the potential relationship between MHC class I molecules and neurogenesis. For the first time, we report the expression......, and we demonstrate they persist in one of the main area of adult neurogenesis, the subventricular zone (SVZ). So far, RAE-1 is only known for its immune functions as a ligand of the activating receptor NKG2D expressed by natural killer (NK) cells, natural killer T, Tγδ, and some T CD8 lymphocytes. Here...... an unexpected role of RAE-1 in regulating adult SVZ neurogenesis by supporting stem/progenitor cells proliferation....

  15. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

    Science.gov (United States)

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-05-26

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

  16. Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

    Science.gov (United States)

    Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

    2014-05-16

    Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

  17. SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer.

    Science.gov (United States)

    Papadakis, Andreas I; Sun, Chong; Knijnenburg, Theo A; Xue, Yibo; Grernrum, Wipawadee; Hölzel, Michael; Nijkamp, Wouter; Wessels, Lodewyk F A; Beijersbergen, Roderick L; Bernards, Rene; Huang, Sidong

    2015-04-01

    Recurrent inactivating mutations in components of SWI/SNF chromatin-remodeling complexes have been identified across cancer types, supporting their roles as tumor suppressors in modulating oncogenic signaling pathways. We report here that SMARCE1 loss induces EGFR expression and confers resistance to MET and ALK inhibitors in non-small cell lung cancers (NSCLCs). We found that SMARCE1 binds to regulatory regions of the EGFR locus and suppresses EGFR transcription in part through regulating expression of Polycomb Repressive Complex component CBX2. Addition of the EGFR inhibitor gefitinib restores the sensitivity of SMARCE1-knockdown cells to MET and ALK inhibitors in NSCLCs. Our findings link SMARCE1 to EGFR oncogenic signaling and suggest targeted treatment options for SMARCE1-deficient tumors.

  18. Shifting boundaries of retinoic acid activity control hindbrain segmental gene expression.

    Science.gov (United States)

    Sirbu, Ioan Ovidiu; Gresh, Lionel; Barra, Jacqueline; Duester, Gregg

    2005-06-01

    Retinoic acid (RA) generated by Raldh2 in paraxial mesoderm is required for specification of the posterior hindbrain, including restriction of Hoxb1 expression to presumptive rhombomere 4 (r4). Hoxb1 expression requires 3' and 5' RA response elements for widespread induction up to r4 and for r3/r5 repression, but RA has previously been detected only from r5-r8, and vHnf1 is required for repression of Hoxb1 posterior to r4 in zebrafish. We demonstrate in mouse embryos that an RA signal initially travels from the paraxial mesoderm to r3, forming a boundary next to the r2 expression domain of Cyp26a1 (which encodes an RA-degrading enzyme). After Hoxb1 induction, the RA boundary quickly shifts to r4/r5, coincident with induction of Cyp26c1 in r4. A functional role for Cyp26c1 in RA degradation was established through examination of RA-treated embryos. Analysis of Raldh2-/- and vHnf1-/- embryos supports a direct role for RA in Hoxb1 induction up to r4 and repression in r3/r5, as well as an indirect role for RA in Hoxb1 repression posterior to r4 via RA induction of vHnf1 up to the r4/r5 boundary. Our findings suggest that Raldh2 and Cyp26 generate shifting boundaries of RA activity, such that r3-r4 receives a short pulse of RA and r5-r8 receives a long pulse of RA. These two pulses of RA activity function to establish expression of Hoxb1 and vHnf1 on opposite sides of the r4/r5 boundary.

  19. Klumpfuss controls FMRFamide expression by enabling BMP signaling within the NB5-6 lineage

    OpenAIRE

    Losada-Perez, Maria; Gabilondo, Hugo; Molina, Isabel; Turiegano, Enrique; Torroja, Laura; Thor, Stefan; Benito-Sipos, Jonathan

    2013-01-01

    A number of transcription factors that are expressed within most, if not all, embryonic neuroblast (NB) lineages participate in neural subtype specification. Some have been extensively studied in several NB lineages (e.g. components of the temporal gene cascade) whereas others only within specific NB lineages. To what extent they function in other lineages remains unknown. Klumpfuss (Klu), the Drosophila ortholog of the mammalian Wilms tumor 1 (WT1) protein, is one such transcription factor. ...

  20. Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

    OpenAIRE

    1986-01-01

    Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting th...

  1. Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis

    OpenAIRE

    Xie, Liqiong; Yang, Cangjing; Wang, Xuelu

    2011-01-01

    The phytohormones, brassinosteroids (BRs), play important roles in regulating cell elongation and cell size, and BR-related mutants in Arabidopsis display significant dwarf phenotypes. Cellulose is a biopolymer which has a major contribution to cell wall formation during cell expansion and elongation. However, whether BRs regulate cellulose synthesis, and if so, what the underlying mechanism of cell elongation induced by BRs is, is unknown. The content of cellulose and the expression levels o...

  2. Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    OpenAIRE

    Natarajan, Kartiga; Gottipati, Koteswara R.; Berhane, Kiflu; Samten, Buka; Pendurthi, Usha; Boggaram, Vijay

    2016-01-01

    Background Persistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial ce...

  3. Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Hecht, Emelia [Department of Medicine, McGill University, Montreal, Quebec (Canada); Zago, Michela [Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Sarill, Miles [Department of Medicine, McGill University, Montreal, Quebec (Canada); Rico de Souza, Angela [Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Gomez, Alvin; Matthews, Jason [Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON (Canada); Hamid, Qutayba; Eidelman, David H. [Department of Medicine, McGill University, Montreal, Quebec (Canada); Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Baglole, Carolyn J., E-mail: Carolyn.baglole@McGill.ca [Department of Medicine, McGill University, Montreal, Quebec (Canada); Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

    2014-11-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR{sup −/−}) and wild-type (AhR{sup +/+}) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR{sup −/−} cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR{sup −/−} compared to AhR{sup +/+} cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR{sup +/+} fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR{sup +/+} lung fibroblasts in response to serum, corresponding to a decrease in p27{sup KIP1} protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27{sup KIP1} in AhR{sup −/−} fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates the

  4. Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis.

    Science.gov (United States)

    Xie, Liqiong; Yang, Cangjing; Wang, Xuelu

    2011-08-01

    The phytohormones, brassinosteroids (BRs), play important roles in regulating cell elongation and cell size, and BR-related mutants in Arabidopsis display significant dwarf phenotypes. Cellulose is a biopolymer which has a major contribution to cell wall formation during cell expansion and elongation. However, whether BRs regulate cellulose synthesis, and if so, what the underlying mechanism of cell elongation induced by BRs is, is unknown. The content of cellulose and the expression levels of the cellulose synthase genes (CESAs) was measured in BR-related mutants and their wild-type counterpart. The chromatin immunoprecipitation (CHIP) experiments and genetic analysis were used to demonstrate that BRs regulate CESA genes. It was found here that the BR-deficient or BR-perceptional mutants contain less cellulose than the wild type. The expression of CESA genes, especially those related to primary cell wall synthesis, was reduced in det2-1 and bri1-301, and was only inducible by BRs in the BR-deficient mutant det2-1. CHIP experiments show that the BR-activated transcription factor BES1 can associate with upstream elements of most CESA genes particularly those related with the primary cell wall. Furthermore, over-expression of the BR receptor BRI1 in CESA1, 3, and 6 mutants can only partially rescue the dwarf phenotypes. Our findings provide potential insights into the mechanism that BRs regulate cellulose synthesis to accomplish the cell elongation process in plant development.

  5. Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

    Directory of Open Access Journals (Sweden)

    Diego Balboa

    2015-09-01

    Full Text Available CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.

  6. Rice zinc finger protein DST enhances grain production through controlling Gn1a/OsCKX2 expression.

    Science.gov (United States)

    Li, Shuyu; Zhao, Bingran; Yuan, Dingyang; Duan, Meijuan; Qian, Qian; Tang, Li; Wang, Bao; Liu, Xiaoqiang; Zhang, Jie; Wang, Jun; Sun, Jiaqiang; Liu, Zhao; Feng, Yu-Qi; Yuan, Longping; Li, Chuanyou

    2013-02-19

    The phytohormone cytokinin (CK) positively regulates the activity and function of the shoot apical meristem (SAM), which is a major parameter determining seed production. The rice (Oryza sativa L.) Gn1a/OsCKX2 (Grain number 1a/Cytokinin oxidase 2) gene, which encodes a cytokinin oxidase, has been identified as a major quantitative trait locus contributing to grain number improvement in rice breeding practice. However, the molecular mechanism of how the expression of OsCKX2 is regulated in planta remains elusive. Here, we report that the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) directly regulates OsCKX2 expression in the reproductive meristem. DST-directed expression of OsCKX2 regulates CK accumulation in the SAM and, therefore, controls the number of the reproductive organs. We identify that DST(reg1), a semidominant allele of the DST gene, perturbs DST-directed regulation of OsCKX2 expression and elevates CK levels in the reproductive SAM, leading to increased meristem activity, enhanced panicle branching, and a consequent increase of grain number. Importantly, the DST(reg1) allele provides an approach to pyramid the Gn1a-dependent and Gn1a-independent effects on grain production. Our study reveals that, as a unique regulator of reproductive meristem activity, DST may be explored to facilitate the genetic enhancement of grain production in rice and other small grain cereals.

  7. Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas.

    Science.gov (United States)

    Drapier, D.; Girard-Bascou, J.; Wollman, F. A.

    1992-03-01

    We analyzed three nuclear mutants of Chlamydomonas reinhardtii altered in the expression of the chloroplast genes atpA or atpB coding for the [alpha] or [beta] subunit of the chloroplast ATP synthase. These mutants revealed the existence of three nuclear products controlling the expression of the two chloroplast genes: the first one acts on the translation of the atpA transcript, and the two others act specifically on the stability of either the atpB or the atpA mRNAs. The nuclear mutation responsible for the decreased stability of the atpB mRNA prevented translation of the corresponding polypeptide. In contrast, the mutation responsible for the decreased stability of the atpA mRNA had limited effect on the translation of the [alpha] subunit, thereby allowing its accumulation and assembly in an active ATP synthase. Although acting originally on the expression of only one of the two main coupling factor 1 subunits, the three mutations caused a change in the translation rate of the other subunit, as viewed in 5-min pulse labeling experiments. This is indicative of a concerted expression of the [alpha] and [beta] subunits at an early post-translational step, or during translation, that may be critical for the assembly of the chloroplast ATP synthase.

  8. Antisense RNA controls LRP1 Sense transcript expression through interaction with a chromatin-associated protein, HMGB2.

    Science.gov (United States)

    Yamanaka, Yasunari; Faghihi, Mohammad Ali; Magistri, Marco; Alvarez-Garcia, Oscar; Lotz, Martin; Wahlestedt, Claes

    2015-05-12

    Long non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer's disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

  9. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

    Science.gov (United States)

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L

    2012-08-01

    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  10. Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

    Directory of Open Access Journals (Sweden)

    Yasunari Yamanaka

    2015-05-01

    Full Text Available Long non-coding RNAs (lncRNAs, including natural antisense transcripts (NATs, are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1, referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2 and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer’s disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

  11. The facial neural crest controls fore- and midbrain patterning by regulating Foxg1 expression through Smad1 activity.

    Science.gov (United States)

    Aguiar, Diego P; Sghari, Soufien; Creuzet, Sophie

    2014-06-01

    The facial neural crest (FNC), a pluripotent embryonic structure forming craniofacial structures, controls the activity of brain organisers and stimulates cerebrum growth. To understand how the FNC conveys its trophic effect, we have studied the role of Smad1, which encodes an intracellular transducer, to which multiple signalling pathways converge, in the regulation of Foxg1. Foxg1 is a transcription factor essential for telencephalic specification, the mutation of which leads to microcephaly and mental retardation. Smad1 silencing, based on RNA interference (RNAi), was performed in pre-migratory FNC cells. Soon after electroporation of RNAi molecules, Smad1 inactivation abolished the expression of Foxg1 in the chick telencephalon, resulting in dramatic microcephaly and partial holoprosencephaly. In addition, the depletion of Foxg1 activity altered the expression Otx2 and Foxa2 in di/mesencephalic neuroepithelium. However, when mutated forms of Smad1 mediating Fgf and Wnt signalling were transfected into FNC cells, these defects were overcome. We also show that, downstream of Smad1 activity, Dkk1, a Wnt antagonist produced by the FNC, initiated the specification of the telencephalon by regulating Foxg1 activity. Additionally, the activity of Cerberus in FNC-derived mesenchyme synergised with Dkk1 to control Foxg1 expression and maintain the balance between Otx2 and Foxa2.

  12. Empathy and recognition of facial expressions of emotion in sex offenders, non-sex offenders and normal controls.

    Science.gov (United States)

    Gery, Isabelle; Miljkovitch, Raphaële; Berthoz, Sylvie; Soussignan, Robert

    2009-02-28

    Research conducted on empathy and emotional recognition in sex offenders is contradictory. The present study was aimed to clarify this issue by controlling for some affective and social variables (depression, anxiety, and social desirability) that are presumed to influence emotional and empathic measures, using a staged multicomponent model of empathy. Incarcerated sex offenders (child molesters), incarcerated non-sex offenders, and non-offender controls (matched for age, gender, and education level) performed a recognition task of facial expressions of basic emotions that varied in intensity, and completed various self-rating scales designed to assess distinct components of empathy (perspective taking, affective empathy, empathy concern, and personal distress), as well as depression, anxiety, and social desirability. Sex offenders were less accurate than the other participants in recognizing facial expressions of anger, disgust, surprise and fear, with problems in confusing fear with surprise, and disgust with anger. Affective empathy was the only component that discriminated sex offenders from non-sex offenders and was correlated with accuracy recognition of emotional expressions. Although our findings must be replicated with a larger number of participants, they support the view that sex offenders might have impairments in the decoding of some emotional cues conveyed by the conspecifics' face, which could have an impact on affective empathy.

  13. Estrogens maintain bone mass by regulating expression of genes controlling function and life span in mature osteoclasts.

    Science.gov (United States)

    Imai, Yuuki; Youn, Ming-Young; Kondoh, Shino; Nakamura, Takashi; Kouzmenko, Alexander; Matsumoto, Takahiro; Takada, Ichiro; Takaoka, Kunio; Kato, Shigeaki

    2009-09-01

    Estrogens play a key role in regulation of bone mass and strength by controlling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. Cellular effects of estrogens are mediated predominantly by the action of estrogen receptor alpha (ERalpha). In earlier studies, ablation of the ERalpha gene in mice did not result in osteoporotic phenotypes due to systemic endocrine disturbance and compensatory effects of elevated levels of testosterone. Despite the relatively well-established effects in osteoblasts, little is known about the direct action of estrogen in osteoclasts. Development in the last decade of more sophisticated genetic manipulation approaches opened new possibilities to explore cell-specific roles of nuclear receptors in bone tissue. Recently, we have generated osteoclast-specific ERalpha gene knockout mice and shown that in vivo estrogens directly regulate the life span of mature osteoclasts by inducing the expression of pro-apoptotic Fas ligand (FasL). Inhibitory effects of estrogens on osteoclast function were further studied in vitro. We observed sufficiently detectable ERalpha expression in osteoclasts differentiating from primary bone marrow cells or RAW264 cells, although levels of ERalpha were decreasing during progression of the differentiation into mature osteoclasts. Treatment with estrogens led to reduction in expression of osteoclast-specific genes controlling bone resorption activity. However, estrogens did not affect the size of multinucleated osteoclasts or number of nuclei in a mature osteoclast. In conclusion, in osteoclasts, estrogens function to inhibit bone resorption activity and vitality rather than differentiation.

  14. Myocardial chemokine expression and intensity of myocarditis in Chagas cardiomyopathy are controlled by polymorphisms in CXCL9 and CXCL10.

    Directory of Open Access Journals (Sweden)

    Luciana Gabriel Nogueira

    Full Text Available BACKGROUND: Chronic Chagas cardiomyopathy (CCC, a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium. METHODS AND RESULTS: Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2-6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes. CONCLUSIONS: Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our

  15. Reduced mRNA expression of PTGDS in peripheral blood mononuclear cells of rapid-cycling bipolar disorder patients compared with healthy control subjects

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Peijs, Lone; Kessing, Lars Vedel;

    2015-01-01

    that mRNA expression of PTGDS and AKR1C3 is deregulated in rapid-cycling disorder patients in a euthymic or current affective state compared with healthy control subjects, and that expression alters with affective states. METHODS: PTGDS and AKR1C3 mRNA expression in peripheral blood mononuclear cells...... was measured in 37 rapid-cycling bipolar disorder patients and 40 age- and gender-matched healthy control subjects using reverse transcription quantitative real-time polymerase chain reaction. Repeated measurements of PTGDS and AKR1C3 mRNA expression were obtained in various affective states during 6-12 months...

  16. Karyopherin alpha2: a control step of glucose-sensitive gene expression in hepatic cells.

    Science.gov (United States)

    Guillemain, Ghislaine; Muñoz-Alonso, Maria J; Cassany, Aurélia; Loizeau, Martine; Faussat, Anne-Marie; Burnol, Anne-Françoise; Leturque, Armelle

    2002-05-15

    Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

  17. Splice Isoforms of Phosducin-like Protein Control the Expression of Heterotrimeric G Proteins*

    Science.gov (United States)

    Gao, Xueli; Sinha, Satyabrata; Belcastro, Marycharmain; Woodard, Catherine; Ramamurthy, Visvanathan; Stoilov, Peter; Sokolov, Maxim

    2013-01-01

    Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein β subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of Gβ and the assembly of Gβ and Gγ subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent Gβ. As a result, the cellular levels of Gβ and Gγ, which depends on Gβ for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the Gα transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins. PMID:23888055

  18. The methods to generate transgenic animals and to control transgene expression.

    Science.gov (United States)

    Houdebine, Louis-Marie

    2002-09-25

    Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy. Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

  19. Staphylococcus aureus ArcR controls expression of the arginine deiminase operon.

    Science.gov (United States)

    Makhlin, Julia; Kofman, Tzili; Borovok, Ilya; Kohler, Christian; Engelmann, Susanne; Cohen, Gerald; Aharonowitz, Yair

    2007-08-01

    We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

  20. Dietary approaches to stop hypertension influence on insulin receptor substrate-1gene expression: A randomized controlled clinical trial

    Directory of Open Access Journals (Sweden)

    Marzieh Kafeshani

    2015-01-01

    Full Text Available Background: Insulin receptor substrate (IRS Type 1 is a main substrate for the insulin receptor, controls insulin signaling in skeletal muscle, adipose tissue, and the vascular, so it is an important candidate gene for insulin resistance (IR. We aimed to compare the effects of the Dietary Approaches to Stop Hypertension (DASH and Usual Dietary Advices (UDA on IRS1 gene expression in women at risk for cardiovascular disease. Materials and Methods: A randomized controlled clinical trial was performed in 44 women at risk for cardiovascular disease. Participants were randomly assigned to a UDA diet or the DASH diet. The DASH diet was rich in fruits, vegetables, whole grains, and low-fat dairy products and low in saturated fat, total fat, cholesterol, refined grains, and sweets, with a total of 2400 mg/day sodium. The UDA diet was a regular diet with healthy dietary advice. Gene expression was assessed by the real-time polymerase chain reaction at the first of study and after 12 weeks. Independent sample t-test and paired-samples t-test were used to compare means of all variables within and between two groups respectively. Results: IRS1 gene expression was increased in DASH group compared with UDA diet (P = 0.00. Weight and waist circumference decreased in DASH group significantly compared to the UDA group (P < 0.05 but the results between the two groups showed no significant difference. Conclusion: DASH diet increased IRS1 gene expression and probably has beneficial effects on IR risks.

  1. Zinc transporter gene expression and glycemic control in post-menopausal women with Type 2 diabetes mellitus.

    Science.gov (United States)

    Foster, Meika; Chu, Anna; Petocz, Peter; Samman, Samir

    2014-10-01

    Type 2 diabetes mellitus (DM) is associated often with underlying zinc deficiency and nutritional supplements such as zinc may be of therapeutic benefit in the disease. In a randomized, double-blind, placebo-controlled, 12-week trial in postmenopausal women (n=48) with Type 2 DM we investigated the effects of supplementation with zinc (40mg/d) and flaxseed oil (FSO; 2g/d) on the gene expression of zinc transporters (ZnT1, ZnT5, ZnT6, ZnT7, ZnT8, Zip1, Zip3, Zip7, and Zip10) and metallothionein (MT-1A, and MT-2A), and markers of glycemic control (glucose, insulin, glycosylated hemoglobin [HbA1c]). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. No significant effects of zinc or FSO supplementation were observed on glycemic marker concentrations, HOMA-IR or fold change over 12 weeks in zinc transporter and metallothionein gene expression. In multivariate analysis, the change over 12 weeks in serum glucose concentrations (P=0.001) and HOMA-IR (P=0.001) predicted the fold change in Zip10. In secondary analysis, marginal statistical significance was observed with the change in both serum glucose concentrations (P=0.003) and HOMA-IR (P=0.007) being predictive of the fold change in ZnT6. ZnT8 mRNA expression was variable; HbA1c levels were higher (P=0.006) in participants who exhibited ZnT8 expression compared to those who did not. The significant predictive relationships between Zip10, ZnT6, serum glucose and HOMA-IR are preliminary, as is the relationship between HbA1c and ZnT8; nevertheless the observations support an association between Type 2 DM and zinc homeostasis that requires further exploration.

  2. Trace metals and over-expression of metallothioneins in bladder tumoral lesions: a case-control study

    Directory of Open Access Journals (Sweden)

    Cymbron Teresa

    2009-07-01

    Full Text Available Abstract Background Previous studies have provided some evidence of a possible association between cancer and metallothioneins. Whether this relates to an exposure to carcinogenic metals remains unclear. Methods In order to examine the association between the expression of metallothioneins and bladder tumors, and to compare the levels of arsenic, cadmium, chromium, lead and nickel in animals with bladder tumors and animals without bladder tumors, 37 cases of bovine bladder tumors and 17 controls were collected. The detection and quantification of metallothioneins in bladder tissue of both cases and controls was performed by immunohistochemistry. And the quantification of metals in tissue and hair was assessed by inductively coupled plasma – mass spectrometry. Results Increased expression of metallothioneins was associated with bladder tumors when compared with non-tumoral bladder tissue (OR = 9.3, 95% CI: 1.0 – 480. The concentrations of cadmium, chromium, lead and nickel in hair of cases were significantly higher than those of controls. However, as for the concentration of metals in bladder tissue, the differences were not significant. Conclusion Though the sample size was small, the present study shows an association between bladder tumors and metallothioneins. Moreover, it shows that concentrations of metals such as cadmium, chromium, lead and nickel in hair may be used as a biomarker of exposure.

  3. Nutritional control of gene expression in Drosophila larvae via TOR, Myc and a novel cis-regulatory element

    Directory of Open Access Journals (Sweden)

    Grewal Savraj S

    2010-01-01

    Full Text Available Abstract Background Nutrient availability is a key determinant of eukaryotic cell growth. In unicellular organisms many signaling and transcriptional networks link nutrient availability to the expression of metabolic genes required for growth. However, less is known about the corresponding mechanisms that operate in metazoans. We used gene expression profiling to explore this issue in developing Drosophila larvae. Results We found that starvation for dietary amino acids (AA's leads to dynamic changes in transcript levels of many metabolic genes. The conserved insulin/PI3K and TOR signaling pathways mediate nutrition-dependent growth in Drosophila and other animals. We found that many AA starvation-responsive transcripts were also altered in TOR mutants. In contrast, although PI3K overexpression induced robust changes in the expression of many metabolic genes, these changes showed limited overlap with the AA starvation expression profile. We did however identify a strong overlap between genes regulated by the transcription factor, Myc, and AA starvation-responsive genes, particularly those involved in ribosome biogenesis, protein synthesis and mitochondrial function. The consensus Myc DNA binding site is enriched in promoters of these AA starvation genes, and we found that Myc overexpression could bypass dietary AA to induce expression of these genes. We also identified another sequence motif (Motif 1 enriched in the promoters of AA starvation-responsive genes. We showed that Motif 1 was both necessary and sufficient to mediate transcriptional responses to dietary AA in larvae. Conclusions Our data suggest that many of the transcriptional effects of amino acids are mediated via signaling through the TOR pathway in Drosophila larvae. We also find that these transcriptional effects are mediated through at least two mechanisms: via the transcription factor Myc, and via the Motif 1 cis-regulatory element. These studies begin to elucidate a nutrient

  4. Post-transcriptional control of DGCR8 expression by the Microprocessor.

    Science.gov (United States)

    Triboulet, Robinson; Chang, Hao-Ming; Lapierre, Robert J; Gregory, Richard I

    2009-06-01

    The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.

  5. The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter

    Institute of Scientific and Technical Information of China (English)

    WangHao; BaiYongyan

    1990-01-01

    The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expression vector which can be used in Ti plasmid system of Agrobacterium iumefaciens.The original plasmid,which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facilitate the selection of recombinant with Ti plasmid.Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter.This chimaeric gene was introduced into integrative Ti plasmid vector pGV 3850,and then transformed into Nicotiana tobaccum the chimaeric gene into tobacco cells.In both cases,the expression of ocs gene was demonstrated.The amount of octopine was much more than the nopaline synthesized by nopaline synthase (nos) gene transferred at the same time with Ti plasmid vector.This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the promoter of nos in tobacco cells.

  6. Angiotensinogen gene expression in adipose tissue: analysis of obese models and hormonal and nutritional control.

    Science.gov (United States)

    Jones, B H; Standridge, M K; Taylor, J W; Moustaïd, N

    1997-07-01

    Synthesis of angiotensin II (ANG II) has recently been described in adipose cells and has been linked to regulation of adiposity. Angiotensinogen (AGT), the substrate from which ANG II is formed, was previously shown to be elevated in adipose tissue of obese (ob/ob and db/db) mice and regulated by nutritional manipulation. It is unknown, however, whether overexpression of adipose AGT can be extended to other models of obesity and whether hormonal and/or nutritional factors directly regulate AGT expression in adipocytes. We investigated these possibilities by analyzing AGT mRNA levels in adipose tissue of obese Zucker rats, viable yellow (Avy) mice, and humans and by treating 3T3-L1 adipocytes with insulin, glucose, and a beta-adrenergic agonist. We demonstrate that AGT mRNA is decreased by approximately 50 and 80%, respectively, in adipose tissue of obese vs. lean Zucker rats and Avy mice. We also report that AGT is expressed at variable levels in human adipose tissue. Finally, we show that AGT mRNA is upregulated by insulin and downregulated by beta-adrenergic stimulation in adipocytes.

  7. The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Brian P Dilkes

    2008-12-01

    Full Text Available The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1, was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring.

  8. TNF-α-Induced microRNAs Control Dystrophin Expression in Becker Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Alyson A. Fiorillo

    2015-09-01

    Full Text Available The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45–47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven microRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31. microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping-treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNF-α increases microRNA levels in vitro whereas NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.

  9. Protruding structures on caterpillars are controlled by ectopic Wnt1 expression.

    Directory of Open Access Journals (Sweden)

    Mina Edayoshi

    Full Text Available Spine-like or protruding structures, which may be aposematic for predators, are often observed in multiple segments of lepidopteran larvae (caterpillars. For example, the larvae of the Chinese wheel butterfly, Byasa alcinous, display many protrusions on their backs as a warning that they are toxic. Although these protrusions are formed by an integument lined with single-layered epidermal cells, the molecular mechanisms underlying their formation have remained unclear. In this study, we focused on a spontaneous mutant of the silkworm, Bombyx mori, Knobbed, which shows similar protrusions to B. alcinous and demonstrates that Wnt1 plays a crucial role in the formation of protrusion structures. Using both transgene expression and RNAi-based knockdown approaches, we showed that Wnt1 designates the position where epidermal cells excessively proliferate, leading to the generation of knobbed structures. Furthermore, in the B. alcinous larvae, Wnt1 was also specifically expressed in association with the protrusions. Our results suggest that Wnt1 plays a role in the formation of protrusions on the larval body, and is conserved broadly among diverse species in Lepidoptera.

  10. Nodal promotes mir206 expression to control convergence and extension movements during zebrafish gastrulation.

    Science.gov (United States)

    Liu, Xiuli; Ma, Yuanqing; Zhang, Congwei; Wei, Shi; Cao, Yu; Wang, Qiang

    2013-10-20

    Nodal, a member of the transforming growth factor β (TGF-β) superfamily, has been shown to play a role in mesendoderm induction and gastrulation movements. The activity of Nodal signaling can be modulated by microRNAs (miRNAs) as previously reported, but little is known about which miRNAs are regulated by Nodal during gastrulation. In the present study, we found that the expression of mir206, one of the most abundant miRNAs during zebrafish early embryo development, is regulated by Nodal signaling. Abrogation of Nodal signal activity results in defective convergence and extension (CE) movements, and these cell migration defects can be rescued by supplying an excess of mir206, suggesting that mir206 acts downstream of Nodal signaling to regulate CE movements. Furthermore, in mir206 morphants, the expression of cell adhesion molecule E-cadherin is significantly increased, while the key transcriptional repressor of E-cadherin, snail1a, is depressed. Our study uncovers a novel mechanism by which Nodal-regulated mir206 modulates gastrulation movements in connection with the Snail/E-cadherin pathway.

  11. Prefrontal NMDA receptors expressed in excitatory neurons control fear discrimination and fear extinction.

    Science.gov (United States)

    Vieira, Philip A; Corches, Alex; Lovelace, Jonathan W; Westbrook, Kevin B; Mendoza, Michael; Korzus, Edward

    2015-03-01

    N-methyl-D-aspartate receptors (NMDARs) are critically involved in various learning mechanisms including modulation of fear memory, brain development and brain disorders. While NMDARs mediate opposite effects on medial prefrontal cortex (mPFC) interneurons and excitatory neurons, NMDAR antagonists trigger profound cortical activation. The objectives of the present study were to determine the involvement of NMDARs expressed specifically in excitatory neurons in mPFC-dependent adaptive behaviors, specifically fear discrimination and fear extinction. To achieve this, we tested mice with locally deleted Grin1 gene encoding the obligatory NR1 subunit of the NMDAR from prefrontal CamKIIα positive neurons for their ability to distinguish frequency modulated (FM) tones in fear discrimination test. We demonstrated that NMDAR-dependent signaling in the mPFC is critical for effective fear discrimination following initial generalization of conditioned fear. While mice with deficient NMDARs in prefrontal excitatory neurons maintain normal responses to a dangerous fear-conditioned stimulus, they exhibit abnormal generalization decrement. These studies provide evidence that NMDAR-dependent neural signaling in the mPFC is a component of a neural mechanism for disambiguating the meaning of fear signals and supports discriminative fear learning by retaining proper gating information, viz. both dangerous and harmless cues. We also found that selective deletion of NMDARs from excitatory neurons in the mPFC leads to a deficit in fear extinction of auditory conditioned stimuli. These studies suggest that prefrontal NMDARs expressed in excitatory neurons are involved in adaptive behavior.

  12. Dynamic Long-Range Chromatin Interaction Controls Expression of IL-21 in CD4+ T Cells.

    Science.gov (United States)

    Park, Joo-Hong; Choi, Yeeun; Song, Min-Ji; Park, Keunhee; Lee, Jong-Joo; Kim, Hyoung-Pyo

    2016-05-15

    IL-21, a pleiotropic cytokine strongly linked with autoimmunity and inflammation, regulates diverse immune responses. IL-21 can be potently induced in CD4(+) T cells by IL-6; however, very little is known about the mechanisms underlying the transcriptional regulation of the Il21 gene at the chromatin level. In this study, we demonstrated that a conserved noncoding sequence located 49 kb upstream of the Il21 gene contains an enhancer element that can upregulate Il21 gene expression in a STAT3- and NFAT-dependent manner. Additionally, we identified enhancer-blocking insulator elements in the Il21 locus, which constitutively bind CTCF and cohesin. In naive CD4(+) T cells, these upstream and downstream CTCF binding sites interact with each other to make a DNA loop; however, the Il21 promoter does not interact with any cis-elements in the Il21 locus. In contrast, stimulation of CD4(+) T cells with IL-6 leads to recruitment of STAT3 to the promoter and novel distal enhancer region. This induces dynamic changes in chromatin configuration, bringing the promoter and the regulatory elements in close spatial proximity. The long-range interaction between the promoter and distal enhancer region was dependent on IL-6/STAT3 signaling pathway but was disrupted in regulatory T cells, where IL-21 expression was repressed. Thus, our work uncovers a novel topological chromatin framework underlying proper transcriptional regulation of the Il21 gene.

  13. Pax6 controls the expression of Lewis x epitope in the embryonic forebrain by regulating alpha 1,3-fucosyltransferase IX expression

    National Research Council Canada - National Science Library

    Shimoda, Yasushi; Tajima, Youichi; Osanai, Taka; Katsume, Asao; Kohara, Michinori; Kudo, Takashi; Narimatsu, Hisashi; Takashima, Noriko; Ishii, Yasuo; Nakamura, Shun; Osumi, Noriko; Sanai, Yutaka

    2002-01-01

    ... cortex as compared with the wild type. Furthermore, expression of a fucosyltransferase gene, FucT-IX, specifically decreased in the mutant, while no change was seen for expression of another fucosyltransferase gene, FucT-IV...

  14. Size-Dependent Expression of the Mitotic Activator Cdc25 Suggests a Mechanism of Size Control in Fission Yeast.

    Science.gov (United States)

    Keifenheim, Daniel; Sun, Xi-Ming; D'Souza, Edridge; Ohira, Makoto J; Magner, Mira; Mayhew, Michael B; Marguerat, Samuel; Rhind, Nicholas

    2017-05-22

    Proper cell size is essential for cellular function. Nonetheless, despite more than 100 years of work on the subject, the mechanisms that maintain cell-size homeostasis are largely mysterious [1]. Cells in growing populations maintain cell size within a narrow range by coordinating growth and division. Bacterial and eukaryotic cells both demonstrate homeostatic size control, which maintains population-level variation in cell size within a certain range and returns the population average to that range if it is perturbed [1, 2]. Recent work has proposed two different strategies for size control: budding yeast has been proposed to use an inhibitor-dilution strategy to regulate size at the G1/S transition [3], whereas bacteria appear to use an adder strategy, in which a fixed amount of growth each generation causes cell size to converge on a stable average [4-6]. Here we present evidence that cell size in the fission yeast Schizosaccharomyces pombe is regulated by a third strategy: the size-dependent expression of the mitotic activator Cdc25. cdc25 transcript levels are regulated such that smaller cells express less Cdc25 and larger cells express more Cdc25, creating an increasing concentration of Cdc25 as cells grow and providing a mechanism for cells to trigger cell division when they reach a threshold concentration of Cdc25. Because regulation of mitotic entry by Cdc25 is well conserved, this mechanism may provide a widespread solution to the problem of size control in eukaryotes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Expression of the lncRNA Maternally Expressed Gene 3 (MEG3) Contributes to the Control of Lung Cancer Cell Proliferation by the Rb Pathway

    OpenAIRE

    2016-01-01

    Maternally expressed gene 3 (MEG3, mouse homolog Gtl2) encodes a long noncoding RNA (lncRNA) that is expressed in many normal tissues, but is suppressed in various cancer cell lines and tumors, suggesting it plays a functional role as a tumor suppressor. Hypermethylation has been shown to contribute to this loss of expression. We now demonstrate that MEG3 expression is regulated by the retinoblastoma protein (Rb) pathway and correlates with a change in cell proliferation. Microarray analysis ...

  16. Elucidation of a novel pathway through which HDAC1 controls cardiomyocyte differentiation through expression of SOX-17 and BMP2.

    Directory of Open Access Journals (Sweden)

    Eneda Hoxha

    Full Text Available Embryonic Stem Cells not only hold a lot of potential for use in regenerative medicine, but also provide an elegant and efficient way to study specific developmental processes and pathways in mammals when whole animal gene knock out experiments fail. We have investigated a pathway through which HDAC1 affects cardiovascular and more specifically cardiomyocyte differentiation in ES cells by controlling expression of SOX17 and BMP2 during early differentiation. This data explains current discrepancies in the role of HDAC1 in cardiovascular differentiation and sheds light into a new pathway through which ES cells determine cardiovascular cell fate.

  17. Evaluation of internal control for gene expression in Phalaenopsis by quantitative real-time PCR.

    Science.gov (United States)

    Yuan, Xiu-Yun; Jiang, Su-Hua; Wang, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2014-07-01

    The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and β-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the

  18. The DNA-binding factor Ctcf critically controls gene expression in macrophages

    NARCIS (Netherlands)

    T. Nikolic (Tatjana); D. Movita (Dowty); M.E.H. Lambers (Margaretha); C. Ribeiro de Almeida (Claudia); P.J. Biesta (Paula); K. Kreefft (Kim); M.J.W. de Bruijn (Marjolein); I.M. Bergen (Ingrid); N.J. Galjart (Niels); P.A. Boonstra (André); R.W. Hendriks (Rudi)

    2014-01-01

    textabstractMacrophages play an important role in immunity and homeostasis. Upon pathogen recognition via specific receptors, they rapidly induce inflammatory responses. This process is tightly controlled at the transcriptional level. The DNA binding zinc-finger protein CCCTC-binding factor (Ctcf) i

  19. A synthetic hybrid promoter for xylose-regulated control of gene expression in Saccharomyces yeasts

    Science.gov (United States)

    Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DN...

  20. Translational control of C/EBPalpha and C/EBPbeta isoform expression

    NARCIS (Netherlands)

    Calkhoven, C F; Müller, C; Leutz, A

    2000-01-01

    Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms re

  1. Indoleamine 2,3-Dioxygenase Is Involved in the Inflammation Response of Corneal Epithelial Cells to Aspergillus fumigatus Infections.

    Directory of Open Access Journals (Sweden)

    Nan Jiang

    Full Text Available Indoleamine 2,3-dioxygenase (IDO, which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs, known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with Aspergillus fumigatus (A. fumigatus and HCECs incubated with spores of A. fumigatus. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by A. fumigatus spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with A. fumigatus stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by A. fumigatus. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to A. fumigatus spores-induced up-regulation of IDO.

  2. Control of response reliability by parvalbumin-expressing interneurons in visual cortex.

    Science.gov (United States)

    Zhu, Yingjie; Qiao, Wenhui; Liu, Kefei; Zhong, Huiyuan; Yao, Haishan

    2015-04-14

    The responses of visual cortical neurons to natural stimuli are both reliable and sparse. These properties require inhibition, yet the contribution of specific types of inhibitory neurons is not well understood. Here we demonstrate that optogenetic suppression of parvalbumin (PV)- but not somatostatin (SOM)-expressing interneurons reduces response reliability in the primary visual cortex of anaesthetized and awake mice. PV suppression leads to increases in the low firing rates and decreases in the high firing rates of cortical neurons, resulting in an overall reduction of the signal-to-noise ratio (SNR). In contrast, SOM suppression generally increases the overall firing rate for most neurons, without affecting the SNR. Further analysis reveals that PV, but not SOM, suppression impairs neural discrimination of natural stimuli. Together, these results reveal a critical role for PV interneurons in the formation of reliable visual cortical representations of natural stimuli.

  3. Retinoid X receptor alpha controls innate inflammatory responses through the up-regulation of chemokine expression.

    Science.gov (United States)

    Núñez, Vanessa; Alameda, Daniel; Rico, Daniel; Mota, Rubén; Gonzalo, Pilar; Cedenilla, Marta; Fischer, Thierry; Boscá, Lisardo; Glass, Christopher K; Arroyo, Alicia G; Ricote, Mercedes

    2010-06-01

    The retinoid X receptor alpha (RXRalpha) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRalpha regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRalpha in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRalpha plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis.

  4. PSD-95 expression controls l-DOPA dyskinesia through dopamine D1 receptor trafficking

    Science.gov (United States)

    Porras, Gregory; Berthet, Amandine; Dehay, Benjamin; Li, Qin; Ladepeche, Laurent; Normand, Elisabeth; Dovero, Sandra; Martinez, Audrey; Doudnikoff, Evelyne; Martin-Négrier, Marie-Laure; Chuan, Qin; Bloch, Bertrand; Choquet, Daniel; Boué-Grabot, Eric; Groc, Laurent; Bezard, Erwan

    2012-01-01

    l-DOPA–induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson’s disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson’s patients. PMID:23041629

  5. Detecting subtle expressions: older adults demonstrate automatic and controlled positive response bias in emotional perception.

    Science.gov (United States)

    Johnson, Dan R; Whiting, Wythe L

    2013-03-01

    The present study examined age differences in emotional perception for the detection of low-intensity, single-emotion facial expressions. Confirming the "positivity effect," at 60 ms and 2,000 ms presentation rates older adults (age = 61+ years, n = 39) exhibited a response bias favoring happy over neutral responses, whereas younger adults (age = 18-23 years, n = 40) favored neutral responses. Furthermore, older adults favored neutral over fearful responses at the 60 ms presentation rate, relative to younger adults. The finding that age differences in response bias were most pronounced at the 60 ms versus 2,000 ms presentation rate suggests that positivity effects in emotional perception rely partly on automatic processing.

  6. Metatranscriptomic insights on gene expression and regulatory controls in Candidatus Accumulibacter phosphatis

    Science.gov (United States)

    Oyserman, Ben O; Noguera, Daniel R; del Rio, Tijana Glavina; Tringe, Susannah G; McMahon, Katherine D

    2016-01-01

    Previous studies on enhanced biological phosphorus removal (EBPR) have focused on reconstructing genomic blueprints for the model polyphosphate-accumulating organism Candidatus Accumulibacter phosphatis. Here, a time series metatranscriptome generated from enrichment cultures of Accumulibacter was used to gain insight into anerobic/aerobic metabolism and regulatory mechanisms within an EBPR cycle. Co-expressed gene clusters were identified displaying ecologically relevant trends consistent with batch cycle phases. Transcripts displaying increased abundance during anerobic acetate contact were functionally enriched in energy production and conversion, including upregulation of both cytoplasmic and membrane-bound hydrogenases demonstrating the importance of transcriptional regulation to manage energy and electron flux during anerobic acetate contact. We hypothesized and demonstrated hydrogen production after anerobic acetate contact, a previously unknown strategy for Accumulibacter to maintain redox balance. Genes involved in anerobic glycine utilization were identified and phosphorus release after anerobic glycine contact demonstrated, suggesting that Accumulibacter routes diverse carbon sources to acetyl-CoA formation via previously unrecognized pathways. A comparative genomics analysis of sequences upstream of co-expressed genes identified two statistically significant putative regulatory motifs. One palindromic motif was identified upstream of genes involved in PHA synthesis and acetate activation and is hypothesized to be a phaR binding site, hence representing a hypothetical PHA modulon. A second motif was identified ~35 base pairs (bp) upstream of a large and diverse array of genes and hence may represent a sigma factor binding site. This analysis provides a basis and framework for further investigations into Accumulibacter metabolism and the reconstruction of regulatory networks in uncultured organisms. PMID:26555245

  7. Regulated expression of virulence gene mviN provides protective immunity and colonization control of Salmonella in poultry.

    Science.gov (United States)

    Rubinelli, Peter M; Lee, Sang In; Roto, Stephanie M; Park, Si Hong; Ricke, Steven C

    2015-10-05

    Current live attenuated vaccines for control of Salmonella in poultry persist in the ceca and may persist in the environment. In this paper we report the construction and characterization of the vaccine efficacy of a Salmonella mutant strain with inducible mviN expression and rapid clearance from the host. The mutant was effective in oral immunization of the broiler chicken host against a virulent Salmonella oral challenge strain, having a mean 7×10(6)CFU/g in the ceca of unvaccinated controls compared to a mean 2×10(3)CFU/g in the ceca of vaccinated chickens at 4 weeks post-challenge (6 weeks of age). The mutant strain also demonstrated immunogenicity, reduced organ colonization, and rapid clearance in broiler chickens within 3 weeks of inoculation.

  8. Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression.

    Science.gov (United States)

    Jacobs, Evan S; Keating, Sheila M; Abdel-Mohsen, Mohamed; Gibb, Stuart L; Heitman, John W; Inglis, Heather C; Martin, Jeffrey N; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C; Young, Mary; Greenblatt, Ruth M; Landay, Alan L; Gange, Stephen J; Deeks, Steven G; Golub, Elizabeth T; Pillai, Satish K; Norris, Philip J

    2017-03-15

    A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4(+) T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4(+) T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4(+) T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the

  9. Reducing CD73 expression by IL1β-Programmed Th17 cells improves immunotherapeutic control of tumors.

    Science.gov (United States)

    Chatterjee, Shilpak; Thyagarajan, Krishnamurthy; Kesarwani, Pravin; Song, Jin H; Soloshchenko, Myroslawa; Fu, Jianing; Bailey, Stefanie R; Vasu, Chenthamarkshan; Kraft, Andrew S; Paulos, Chrystal M; Yu, Xue-Zhong; Mehrotra, Shikhar

    2014-11-01

    T cells of the T helper (Th)17 subset offer promise in adoptive T-cell therapy for cancer. However, current protocols for ex vivo programming of Th17 cells, which include TGFβ exposure, increase the expression of CD39 and CD73, two cell surface ATP ectonucleotidases that reduce T-cell effector functions and promote immunosuppression. Here, we report that ATP-mediated suppression of IFNγ production by Th17 cells can be overcome by genetic ablation of CD73 or by using IL1β instead of TGFβ to program Th17 cells ex vivo. Th17 cells cultured in IL1β were also highly polyfunctional, expressing high levels of effector molecules and exhibiting superior short-term control of melanoma in mice, despite reduced stem cell-like properties. TGFβ addition at low doses that did not upregulate CD73 expression but induced stemness properties drastically improved the antitumor effects of IL1β-cultured Th17 cells. Effector properties of IL1β-dependent Th17 cells were likely related to their high glycolytic capacity, since ex vivo programming in pyruvate impaired glycolysis and antitumor effects. Overall, we show that including TGFβ in ex vivo cultures used to program Th17 cells blunts their immunotherapeutic potential and demonstrate how this potential can be more fully realized for adoptive T-cell therapy.

  10. Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis.

    Science.gov (United States)

    Erickson, David L; Endersby, Ryan; Kirkham, Amanda; Stuber, Kent; Vollman, Dolina D; Rabin, Harvey R; Mitchell, Ian; Storey, Douglas G

    2002-04-01

    Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa. The chronic infections caused by P. aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants, P. aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of N-(3-oxododecanoyl)-L-homoserine (3-oxo-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.

  11. Orthodenticle Is Required for the Expression of Principal Recognition Molecules That Control Axon Targeting in the Drosophila Retina.

    Directory of Open Access Journals (Sweden)

    Chiara Mencarelli

    2015-06-01

    Full Text Available Parallel processing of neuronal inputs relies on assembling neural circuits into distinct synaptic-columns and layers. This is orchestrated by matching recognition molecules between afferent growth cones and target areas. Controlling the expression of these molecules during development is crucial but not well understood. The developing Drosophila visual system is a powerful genetic model for addressing this question. In this model system, the achromatic R1-6 photoreceptors project their axons in the lamina while the R7 and R8 photoreceptors, which are involved in colour detection, project their axons to two distinct synaptic-layers in the medulla. Here we show that the conserved homeodomain transcription factor Orthodenticle (Otd, which in the eye is a main regulator of rhodopsin expression, is also required for R1-6 photoreceptor synaptic-column specific innervation of the lamina. Our data indicate that otd function in these photoreceptors is largely mediated by the recognition molecules flamingo (fmi and golden goal (gogo. In addition, we find that otd regulates synaptic-layer targeting of R8. We demonstrate that during this process, otd and the R8-specific transcription factor senseless/Gfi1 (sens function as independent transcriptional inputs that are required for the expression of fmi, gogo and the adhesion molecule capricious (caps, which govern R8 synaptic-layer targeting. Our work therefore demonstrates that otd is a main component of the gene regulatory network that regulates synaptic-column and layer targeting in the fly visual system.

  12. UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression.

    Science.gov (United States)

    Steber, C M; Esposito, R E

    1995-01-01

    The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes. Images Fig. 3 Fig. 4 Fig. 5 PMID:8618927

  13. Light-controlled modulation of gene expression by chemical optoepigenetic probes.

    Science.gov (United States)

    Reis, Surya A; Ghosh, Balaram; Hendricks, J Adam; Szantai-Kis, D Miklos; Törk, Lisa; Ross, Kenneth N; Lamb, Justin; Read-Button, Willis; Zheng, Baixue; Wang, Hongtao; Salthouse, Christopher; Haggarty, Stephen J; Mazitschek, Ralph

    2016-05-01

    Epigenetic gene regulation is a dynamic process orchestrated by chromatin-modifying enzymes. Many of these master regulators exert their function through covalent modification of DNA and histone proteins. Aberrant epigenetic processes have been implicated in the pathophysiology of multiple human diseases. Small-molecule inhibitors have been essential to advancing our understanding of the underlying molecular mechanisms of epigenetic processes. However, the resolution offered by small molecules is often insufficient to manipulate epigenetic processes with high spatiotemporal control. Here we present a generalizable approach, referred to as 'chemo-optical modulation of epigenetically regulated transcription' (COMET), enabling high-resolution, optical control of epigenetic mechanisms based on photochromic inhibitors of human histone deacetylases using visible light. COMET probes may be translated into new therapeutic strategies for diseases where conditional and selective epigenome modulation is required.

  14. Fish Oil Supplementation and Fatty Acid Synthase Expression in the Prostate: A Randomized Controlled Trial. Addendum

    Science.gov (United States)

    2011-07-01

    acids ( PUFA ), particularly omega -3 fatty acids , inhibits SREBP-1 activation, resulting in a decreased transcription of FAS. 15. SUBJECT TERMS Prostate...Cancer; Lipid Metabolism; Clinical Trial; Omega -3 Fatty Acids 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...controls, Menendez et al demonstrated that addition of omega -3 fatty acids (-3 FA), docosahexanoic acid (DHA), alpha- linolenic acid

  15. Bacteria clustering by polymers induces the expression of quorum sense controlled phenotypes

    OpenAIRE

    Lui, Leong T.; Xue, Xuan; Sui, Cheng; Brown, Alan; Pritchard, David I.; Halliday, Nigel; Winzer, Klaus; Howdle, Stephen M.; Fernandez-Trillo, Francisco; Krasnogor, Natalio; Alexander, Cameron

    2013-01-01

    Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one mean by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are thus a potential means to control bacterial population responses. Here we report how polymeric “bacteria sequestrants”, designed to bind to bacteria through e...

  16. TRAIL based therapy: overview of mesenchymal stem cell based delivery and miRNA controlled expression of TRAIL.

    Science.gov (United States)

    Attar, Rukset; Sajjad, Farhana; Qureshi, Muhammad Zahid; Tahir, Fizza; Hussain, Ejaz; Fayyaz, Sundas; Farooqi, Ammad Ahmad

    2014-01-01

    Rapidly increasing number of outstanding developments in the field of TRAIL mediated signaling have revolutionized our current information about inducing and maximizing TRAIL mediated apoptosis in resistant cancer cells. Data obtained with high-throughput technologies have provided finer resolution of tumor biology and now it is known that a complex structure containing malignant cells strictly coupled with a large variety of surrounding cells constitutes the tumor stroma. Utility of mesenchymal stem cells (MSCs) as cellular vehicles has added new layers of information. There is sufficient experimental evidence substantiating efficient gene deliveries into MSCs by retroviral, lentiviral and adenoviral vectors. Moreover, there is a paradigm shift in molecular oncology and recent high impact research has shown controlled expression of TRAIL in cancer cells on insertion of complementary sequences for frequently downregulated miRNAs. In this review we have attempted to provide an overview of utility of TRAIL engineered MSCs for effective killing of tumor and potential of using miRNA response elements as rheostat like switch to control expression of TRAIL in cancer cells.

  17. A new expression of Ns versus Ef to an accurate control charge model for AlGaAs/GaAs

    Science.gov (United States)

    Bouneb, I.; Kerrour, F.

    2016-03-01

    Semi-conductor components become the privileged support of information and communication, particularly appreciation to the development of the internet. Today, MOS transistors on silicon dominate largely the semi-conductors market, however the diminution of transistors grid length is not enough to enhance the performances and respect Moore law. Particularly, for broadband telecommunications systems, where faster components are required. For this reason, alternative structures proposed like hetero structures IV-IV or III-V [1] have been.The most effective components in this area (High Electron Mobility Transistor: HEMT) on IIIV substrate. This work investigates an approach for contributing to the development of a numerical model based on physical and numerical modelling of the potential at heterostructure in AlGaAs/GaAs interface. We have developed calculation using projective methods allowed the Hamiltonian integration using Green functions in Schrodinger equation, for a rigorous resolution “self coherent” with Poisson equation. A simple analytical approach for charge-control in quantum well region of an AlGaAs/GaAs HEMT structure was presented. A charge-control equation, accounting for a variable average distance of the 2-DEG from the interface was introduced. Our approach which have aim to obtain ns-Vg characteristics is mainly based on: A new linear expression of Fermi-level variation with two-dimensional electron gas density in high electron mobility and also is mainly based on the notion of effective doping and a new expression of AEc

  18. Evening Expression of Arabidopsis GIGANTEA Is Controlled by Combinatorial Interactions among Evolutionarily Conserved Regulatory Motifs[C][W][OPEN

    Science.gov (United States)

    Nordström, Karl; Cremer, Frédéric; Tóth, Réka; Hartke, Martin; Simon, Samson; Klasen, Jonas R.; Bürstel, Ingmar; Coupland, George

    2014-01-01

    Diurnal patterns of gene transcription are often conferred by complex interactions between circadian clock control and acute responses to environmental cues. Arabidopsis thaliana GIGANTEA (GI) contributes to photoperiodic flowering, circadian clock control, and photoreceptor signaling, and its transcription is regulated by the circadian clock and light. We used phylogenetic shadowing to identify three evolutionarily constrained regions (conserved regulatory modules [CRMs]) within the GI promoter and show that CRM2 is sufficient to confer a similar transcriptional pattern as the full-length promoter. Dissection of CRM2 showed that one subfragment (CRM2-A) contributes light inducibility, while another (CRM2-B) exhibits a diurnal response. Mutational analysis showed that three ABA RESPONSE ELEMENT LIKE (ABREL) motifs in CRM2-A and three EVENING ELEMENTs (EEs) in CRM2-B are essential in combination to confer a high amplitude diurnal pattern of expression. Genome-wide analysis identified characteristic spacing patterns of EEs and 71 A. thaliana promoters containing three EEs. Among these promoters, that of FLAVIN BINDING KELCH REPEAT F-BOX1 was analyzed in detail and shown to harbor a CRM functionally related to GI CRM2. Thus, combinatorial interactions among EEs and ABRELs confer diurnal patterns of transcription via an evolutionarily conserved module present in GI and other evening-expressed genes. PMID:25361953

  19. Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage diets

    Science.gov (United States)

    2013-01-01

    Background Both genetic background and finishing system can alter fat deposition, thus indicating their influence on adipogenic and lipogenic factors. However, the molecular mechanisms underlying fat deposition and fatty acid composition in beef cattle are not fully understood. This study aimed to assess the effect of breed and dietary silage level on the expression patterns of key genes controlling lipid metabolism in subcutaneous adipose tissue (SAT) and longissimus lumborum (LL) muscle of cattle. To that purpose, forty bulls from two genetically diverse Portuguese bovine breeds with distinct maturity rates, Alentejana and Barrosã, were selected and fed either low (30% maize silage/70% concentrate) or high silage (70% maize silage/30% concentrate) diets. Results The results suggested that enhanced deposition of fatty acids in the SAT from Barrosã bulls, when compared to Alentejana, could be due to higher expression levels of lipogenesis (SCD and LPL) and β-oxidation (CRAT) related genes. Our results also indicated that SREBF1 expression in the SAT is increased by feeding the low silage diet. Together, these results point out to a higher lipid turnover in the SAT of Barrosã bulls when compared to Alentejana. In turn, lipid deposition in the LL muscle is related to the expression of adipogenic (PPARG and FABP4) and lipogenic (ACACA and SCD) genes. The positive correlation between ACACA expression levels and total lipids, as well trans fatty acids, points to ACACA as a major player in intramuscular deposition in ruminants. Moreover, results reinforce the role of FABP4 in intramuscular fat development and the SAT as the major site for lipid metabolism in ruminants. Conclusions Overall, the results showed that SAT and LL muscle fatty acid composition are mostly dependent on the genetic background. In addition, dietary silage level impacted on muscle lipid metabolism to a greater extent than on that of SAT, as evaluated by gene expression levels of adipogenic and

  20. FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

    Science.gov (United States)

    Gopal, Keshav; Saleme, Bruno; Al Batran, Rami; Aburasayn, Hanin; Eshreif, Amina; Ho, Kim L; Ma, Wayne K; Almutairi, Malak; Eaton, Farah; Gandhi, Manoj; Park, Edwards A; Sutendra, Gopinath; Ussher, John R

    2017-09-01

    Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report

  1. PPARgamma agonism increases rat adipose tissue lipolysis, expression of glyceride lipases, and the response of lipolysis to hormonal control.

    Science.gov (United States)

    Festuccia, W T; Laplante, M; Berthiaume, M; Gélinas, Y; Deshaies, Y

    2006-10-01

    The aim of this study was to investigate the effect and mechanisms of action of in vivo peroxisome proliferator-activated receptor gamma (PPARgamma) activation on white adipose tissue (WAT) lipolysis and NEFA metabolism. Study rats were treated for 7 days with 15 mg/kg of rosiglitazone per day; control rats were not treated. After a 6-h fast, lipolysis and levels of mRNA for lipases were assessed in explants from various adipose depots. Rosiglitazone markedly increased basal and noradrenaline (norepinephrine)-stimulated glycerol and NEFA release from WAT explants, and amplified their inhibition by insulin. Primary adipocytes isolated from PPARgamma agonist-treated rats were also more responsive to noradrenaline stimulation expressed per cell, ruling out a contribution of an altered number of mature adipocytes in explants. Rosiglitazone concomitantly increased levels of mRNA transcripts for adipose triglyceride lipase (ATGL) and monoglyceride lipase (MGL) in subcutaneous and visceral WAT, and mRNA for hormone-sensitive lipase (HSL) in subcutaneous WAT. Lipase expression increased within 12 h of in vitro exposure of naïve explants to rosiglitazone, suggesting direct transcriptional activation. In parallel, chronic in vivo treatment with rosiglitazone lowered plasma NEFAs and in WAT its expected stimulatory action on glycerol and NEFA recycling, and on the expression of genes involved in NEFA uptake and retention by WAT, such processes counteracting net NEFA export. These findings demonstrate that, in the face of its plasma NEFA-lowering action, PPARgamma agonism stimulates WAT lipolysis, an effect that is compensated by lipid-retaining pathways. The results further suggest that PPARgamma agonism stimulates lipolysis by increasing the lipolytic potential, including the expression levels of the genes encoding adipose triglyceride lipase and monoglyceride lipase.

  2. The peroxisome proliferator-activated receptor N-terminal domain controls isotype-selective gene expression and adipogenesis.

    Science.gov (United States)

    Hummasti, Sarah; Tontonoz, Peter

    2006-06-01

    Peroxisome proliferator-activated receptors (PPARgamma, PPARalpha, and PPARdelta) are important regulators of lipid metabolism. Although they share significant structural similarity, the biological effects associated with each PPAR isotype are distinct. For example, PPARalpha and PPARdelta regulate fatty acid catabolism, whereas PPARgamma controls lipid storage and adipogenesis. The different functions of PPARs in vivo can be explained at least in part by the different tissue distributions of the three receptors. The question of whether the receptors have different intrinsic activities and regulate distinct target genes, however, has not been adequately explored. We have engineered cell lines that express comparable amounts of each receptor. Transcriptional profiling of these cells in the presence of selective agonists reveals partially overlapping but distinct patterns of gene regulation by the three PPARs. Moreover, analysis of chimeric receptors points to the N terminus of each receptor as the key determinant of isotype-selective gene expression. For example, the N terminus of PPARgamma confers the ability to promote adipocyte differentiation when fused to the PPARdelta DNA binding domain and ligand binding domain, whereas the N terminus of PPARdelta leads to the inappropriate expression of fatty acid oxidation genes in differentiated adipocytes when fused to PPARgamma. Finally, we demonstrate that the N terminus of each receptor functions in part to limit receptor activity because deletion of the N terminus leads to nonselective activation of target genes. A more detailed understanding of the mechanisms by which the individual PPARs differentially regulate gene expression should aid in the design of more effective drugs, including tissue- and target gene-selective PPAR modulators.

  3. Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M

    2017-08-11

    The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.

    Science.gov (United States)

    Barbagallo, Michael S; Birch, Katherine E; Deacon, Nicholas J; Mosse, Jennifer A

    2012-07-01

    The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.

  5. Chromatin architecture: A new dimension in the dynamic control of gene expression

    KAUST Repository

    Ramirez-Prado, Juan Sebastian

    2016-09-10

    As the most recent evidence of eukaryotic cell complexity, genome architecture has astounded the scientific community and prompted a variety of technical and cognitive challenges. Several technologies have emerged and evidenced the integration of chromatin packaging and topology, epigenetic processes, and transcription for the pertinent regulation of gene expression. In the present addendum we present and discuss some of our recent research, directed toward the holistic comprehension of the processes by which plants respond to environmental and developmental stimuli. We propose that the study of genome topology and genomic interactions is essential for the understanding of the molecular mechanisms behind a phenotype. Even though our knowledge and understanding of genome architecture and hierarchy has improved substantially in the last few years -in Arabidopsis and other eukaryotes -, there is still a long way ahead in this relatively new field of study. For this, it is necessary to take advantage of the high resolution of the emerging available techniques, and perform integrative approaches with which it will be possible to depict the role of chromatin architecture in the regulation of transcription and ultimately, physiological processes.

  6. Control of islet intercellular adhesion molecule-1 expression by interferon-alpha and hypoxia.

    Science.gov (United States)

    Chakrabarti, D; Huang, X; Beck, J; Henrich, J; McFarland, N; James, R F; Stewart, T A

    1996-10-01

    The ability of interferon-alpha (IFN-alpha) to induce the adhesion molecules that characterize the islets of patients with type I diabetes has been investigated. We have found that all tested recombinant IFN-as will induce major histocompatibility complex (MHC) class I on arterial endothelial cells. Some but not all IFN-as will induce intercellular adhesion molecule-1 (ICAM-1). However, there is only a transient and modest increase in VCAM on arterial endothelial cells. IFN-alpha has very little effect on endothelial MHC class II expression but will induce these proteins on monocytes. Thus, there is a close concordance between the biological actions of IFN-alpha and the appearance of those adhesion molecules induced in the islets of patients with type I diabetes. IFN-alpha is also produced in normal human islets during short-term cultures, probably as a result of the ischemia present at the center of the islet. This induction of IFN-alpha by hypoxia may explain the previously reported spontaneous induction of ICAM-1 in human islets and may also be a contributing factor to the failure of islet grafts.

  7. FACT plays a major role in histone dynamics affecting VSG expression site control in Trypanosoma brucei.

    Science.gov (United States)

    Denninger, Viola; Rudenko, Gloria

    2014-11-01

    Chromatin remodelling is involved in the transcriptional regulation of the RNA polymerase I transcribed variant surface glycoprotein (VSG) expression sites (ESs) of Trypanosoma brucei. We show that the T. brucei FACT complex contains the Pob3 and Spt16 subunits, and plays a key role in ES silencing. We see an inverse correlation between transcription and condensed chromatin, whereby FACT knockdown results in ES derepression and more open chromatin around silent ES promoters. Derepressed ESs show increased sensitivity to micrococcal nuclease (MNase) digestion, and a decrease in histones at silent ES promoters but not telomeres. In contrast, FACT knockdown results in more histones at the active ES, correlated with transcription shut-down. ES promoters are derepressed in cells stalled at the G2/M cell cycle stage after knockdown of FACT, but not in G2/M cells stalled after knockdown of cyclin 6. This argues that the observed ES derepression is a direct consequence of histone chaperone activity by FACT at the G2/M cell cycle stage which could affect transcription elongation, rather than an indirect consequence of a cell cycle checkpoint. These experiments highlight the role of the FACT complex in cell cycle-specific chromatin remodelling within VSG ESs. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  8. Expression of the 90K immunostimulator gene is controlled by a promoter with unique features

    DEFF Research Database (Denmark)

    Brakebusch, C; Jallal, B; Fusco, O

    1997-01-01

    90K is a secreted glycoprotein with tumor suppressive functions, which is up-regulated in various types of cancer and in AIDS. In order to understand the regulation of its expression, the mouse 90K gene was isolated and analyzed. The gene spans about 8.8-kilobase pairs and consists of 6 exons...... and was localized on chromosome 11, region E. RNase protection identified one major transcription start site (+1) and three minor ones (-3, +32, +34). The mouse 90K gene was found to have a TATA-less promoter of unusual structure. The 2. 3-kilobase pair 5'-flanking region exhibited strong promoter activity in NIH 3......T3 cells; however, it contained neither a TATA-box nor a SP1 site and was not GC-rich. No known initiator motif was found around the transcription start site. 5'- and 3'-deletions defined a minimal promoter of 51 base pairs (-66 --> -16), not including the start site, essential and sufficient...

  9. Btk expression is controlled by Oct and BOB.1/OBF.1.

    Science.gov (United States)

    Brunner, Cornelia; Wirth, Thomas

    2006-01-01

    BOB.1/OBF.1 is a lymphocyte-restricted transcriptional coactivator. It binds together with the Oct1 and Oct2 transcription factors to DNA and enhances their transactivation potential. Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in differentiation, function and signaling of B cells. In search of BOB.1/OBF.1 regulated genes we identified Btk--a cytoplasmic tyrosine kinase-as a direct target of BOB.1/OBF.1. Analyses of the human as well as murine Btk promoters revealed a non-consensus octamer site close to the start site of transcription. Here we show that Oct proteins together with BOB.1/OBF.1 are able to form ternary complexes on these sites in vitro and in vivo. This in turn leads to the induction of Btk promoter activity in synergism with the transcription factor PU.1. Btk, like BOB.1/OBF.1, plays a critical role in B cell development and B cell receptor signalling. Therefore the down-regulation of Btk expression in BOB.1/OBF.1-deficient B cells could be related to the functional and developmental defects observed in BOB.1/OBF.1-deficient mice.

  10. The intrinsic microglial molecular clock controls synaptic strength via the circadian expression of cathepsin S.

    Science.gov (United States)

    Hayashi, Yoshinori; Koyanagi, Satoru; Kusunose, Naoki; Okada, Ryo; Wu, Zhou; Tozaki-Saitoh, Hidetoshi; Ukai, Kiyoharu; Kohsaka, Shinichi; Inoue, Kazuhide; Ohdo, Shigehiro; Nakanishi, Hiroshi

    2013-09-25

    Microglia are thought to play important roles in the maintenance of neuronal circuitry and the regulation of behavior. We found that the cortical microglia contain an intrinsic molecular clock and exhibit a circadian expression of cathepsin S (CatS), a microglia-specific lysosomal cysteine protease in the brain. The genetic deletion of CatS causes mice to exhibit hyperlocomotor activity and removes diurnal variations in the synaptic activity and spine density of the cortical neurons, which are significantly higher during the dark (waking) phase than the light (sleeping) phase. Furthermore, incubation with recombinant CatS significantly reduced the synaptic activity of the cortical neurons. These results suggest that CatS secreted by microglia during the dark-phase decreases the spine density of the cortical neurons by modifying the perisynaptic environment, leading to downscaling of the synaptic strength during the subsequent light-phase. Disruption of CatS therefore induces hyperlocomotor activity due to failure to downscale the synaptic strength.

  11. Mini Review: Relational Stability in the Expression of Normality, Variation and Control of Thyroid Function

    Directory of Open Access Journals (Sweden)

    Rudolf Hoermann

    2016-11-01

    Full Text Available Thyroid hormone concentrations only become sufficient to maintain a euthyroid state through appropriate stimulation by pituitary TSH. In such a dynamic system under constant high pressure, guarding against overstimulation becomes vital. Therefore, several defensive mechanisms protect against accidental overstimulation, such as plasma protein binding, conversion of T4 into the more active T3, active transmembrane transport, counter-regulatory activities of reverse T3 and thyronamines and negative hypothalamic-pituitary-thyroid feedback control of TSH. TSH has gained a dominant but misguided role in interpreting thyroid function testing in assuming that its exceptional sensitivity thereby translates into superior diagnostic performance. However, TSH-dependent thyroid disease classification is heavily influenced by statistical analytic techniques such as uni- or multivariate-defined normality. This demands a separation of its conjoint roles as a sensitive screening test and accurate diagnostic tool. Homeostatic equilibria (set points in healthy subjects are less variable, and do not follow a pattern of random variation, rather indicating signs of early and progressive homeostatic control across the euthyroid range. In the event of imminent thyroid failure with a reduced FT4 output per unit TSH, conversion efficiency increases in order to maintain FT3 stability. In such situations, T3 stability takes priority over set point maintenance. This suggests a concept of relational stability. These findings have important implications for both TSH reference limits and treatment targets for patients on levothyroxine. The use of archival markers is proposed to facilitate the homeostatic interpretation of all parameters.

  12. Developmental Expression and Glucocorticoid Control of the Leptin Receptor in Fetal Ovine Lung.

    Science.gov (United States)

    De Blasio, Miles J; Boije, Maria; Vaughan, Owen R; Bernstein, Brett S; Davies, Katie L; Plein, Alice; Kempster, Sarah L; Smith, Gordon C S; Charnock-Jones, D Stephen; Blache, Dominique; Wooding, F B Peter; Giussani, Dino A; Fowden, Abigail L; Forhead, Alison J

    2015-01-01

    The effects of endogenous and synthetic glucocorticoids on fetal lung maturation are well-established, although the role of leptin in lung development before birth is unclear. This study examined mRNA and protein levels of the signalling long-form leptin receptor (Ob-Rb) in fetal ovine lungs towards term, and after experimental manipulation of glucocorticoid levels in utero by fetal cortisol infusion or maternal dexamethasone treatment. In fetal ovine lungs, Ob-Rb protein was localised to bronchiolar epithelium, bronchial cartilage, vascular endothelium, alveolar macrophages and type II pneumocytes. Pulmonary Ob-Rb mRNA abundance increased between 100 (0.69 fractional gestational age) and 144 days (0.99) of gestation, and by 2-4-fold in response to fetal cortisol infusion and maternal dexamethasone treatment. In contrast, pulmonary Ob-Rb protein levels decreased near term and were halved by glucocorticoid treatment, without any significant change in phosphorylated signal transducer and activator of transcription-3 (pSTAT3) at Ser727, total STAT3 or the pulmonary pSTAT3:STAT3 ratio. Leptin mRNA was undetectable in fetal ovine lungs at the gestational ages studied. These findings demonstrate differential control of pulmonary Ob-Rb transcript abundance and protein translation, and/or post-translational processing, by glucocorticoids in utero. Localisation of Ob-Rb in the fetal ovine lungs, including alveolar type II pneumocytes, suggests a role for leptin signalling in the control of lung growth and maturation before birth.

  13. Tumor type M2 pyruvate kinase expression in gastric cancer,colorectal cancer and controls

    Institute of Scientific and Technical Information of China (English)

    Bo Zhang; Jian-Ying Chen; Dao-Da Chen; Guo-Bin Wang; Ping Shen

    2004-01-01

    AIM: Tumor formation is generally linked to an expansion of glycolytic phosphometabolite pools and aerobic glycolytic flux rates. To achieve this, tumor cells generally overexpress a special glycolytic isoenzyme, termed pyruvate kinase type M2. The present study was designed to evaluate the use of a new tumor marker, tumor M2-PK, in discriminating gastrointestinal cancer patients from healthy controls, and to compare with the reference tumor markers CEA and CA72-4.METHODS: The concentration of tumor M2-PK in body fluids could be quantitatively determined by a commercially available enzyme-linked immunosorbent assay (ELISA)-kit (ScheBo(R) Tech, Giessen, Germany). By using this kit, the tumor M2-PK concentration was measured in EDTA-plasma of 108 patients. For the healthy blood donors a cut-off value of 15 U/mL was evaluated, which corresponded to 90% specificity. Overall 108 patients were included in this study, 54 patients had a histological confirmed gastric cancer, 54 patients colorectal cancer, and 20 healthy volunteers served as controls.RESULTS: The cut-off value to discriminate patients from controls was established at 15 U/mL for tumor M2-PK. The mean tumor M2-PK concentration of gastric cancer was 26.937 U/mL. According to the TNM stage system, the mean tumor M2-PK concentration of stage Ⅰ was 16.324 U/mL, of stage Ⅱ 15.290 U/mL, of stage Ⅲ 30.289 U/mL, of stage Ⅳ127.31 U/mL, of non-metastasis 12.854 U/mL and of metastasis 35.711 U/mL. The mean Tumor M2-PK concentration of colorectal cancer was 30.588 U/mL. According to the Dukes stage system, the mean tumor M2-PK concentration of Dukes A was 16.638 U/mL, of Dukes B 22.070 U/mL, and of Dukes C 48.024 U/mL, of non-metastasis 19.501 U/mL, of metastasis 49.437 U/mL. The mean tumor M2-PK concentration allowed a significant discrimination of colorectal cancers (30.588 U/mL) from controls (10.965 U/mL) (P<0.01), and gastric cancer (26.937 U/mL) from controls (10.965 U/mL)(P<0.05). The overall

  14. YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells

    Science.gov (United States)

    Poitelon, Y; Lopez-Anido, C; Catignas, K; Berti, C; Palmisano, M; Williamson, C; Ameroso, D; Abiko, K; Hwang, Y; Gregorieff, A; Wrana, J; Asmani, M; Zhao, R; Sim, FJ; Wrabetz, L; Svaren, J; Feltri, ML

    2016-01-01

    Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons, using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice we show that Taz is required in Schwann cells for radial sorting and myelination, and that Yap is redundant with Taz. Yap/Taz are activated in Schwann cells by mechanical stimuli, and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for proper radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells. PMID:27273766

  15. Electronic control of gene expression and cell behaviour in Escherichia coli through redox signalling

    Science.gov (United States)

    Tschirhart, Tanya; Kim, Eunkyoung; McKay, Ryan; Ueda, Hana; Wu, Hsuan-Chen; Pottash, Alex Eli; Zargar, Amin; Negrete, Alejandro; Shiloach, Joseph; Payne, Gregory F.; Bentley, William E.

    2017-01-01

    The ability to interconvert information between electronic and ionic modalities has transformed our ability to record and actuate biological function. Synthetic biology offers the potential to expand communication `bandwidth' by using biomolecules and providing electrochemical access to redox-based cell signals and behaviours. While engineered cells have transmitted molecular information to electronic devices, the potential for bidirectional communication stands largely untapped. Here we present a simple electrogenetic device that uses redox biomolecules to carry electronic information to engineered bacterial cells in order to control transcription from a simple synthetic gene circuit. Electronic actuation of the native transcriptional regulator SoxR and transcription from the PsoxS promoter allows cell response that is quick, reversible and dependent on the amplitude and frequency of the imposed electronic signals. Further, induction of bacterial motility and population based cell-to-cell communication demonstrates the versatility of our approach and potential to drive intricate biological behaviours.